[Histonet] I did it I am now a certified HTL
Hi Everyone After years of putting it off, I finally took my ASCP HTL exam and passed it. Huzzah! Patrick. Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Advice on exam prep for the ASCP HTL exam
Hi everyone I am going to take my ASCP HTL exam on the 21st of April Any advice for studying? I have been going over the sample online tests for both the HT and the HTL and I bought the Michigan study guide. Thanks Patrick Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Problem with ccmount drying out and reexposing tissue sections
Hi everyone, Has anyone experienced a problem with sigmas cc/mount solution. I liberally cover my section with it to form a protective seal., But with my current bottle it seems that when it dries it re-exposes the tissue and does not form a protective layer. I end up having to reapply it. The 2nd apply seems to get me the protective layer that I want, but I am concerned that drying/exposure from the first application will damage the staining that I have. I don't want my post staining slides to be exposed to air for a length of time between hematoxylin and cover slipping. This is a fairly recent problem and I am wondering if I just have a bad batch of cc/mount. CAT# C9368-30 ML Lot# MKBR27838V I called Sigma and they are sending me a new bottle from a different lot. But they haven't experienced this problem before with cc/mount. Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Quick H202 quenching question.
If I have an IHC where I am staining 2 slides from the same block, one with one primary antibody and the other with a different primary antibody. And one antibody's slides have high nonspecific background, but the other antibody's slides have no background, Can I deduce that the background staining is not caused by insufficient H202 quenching/blocking? All the steps were the same, except for the primary antibody/secondary antibody. Also, they did have different epitope retrievals. But the wash buffers/blocking buffers/and substrate were the same. I am fairly confident that my background problems are related to this particular primary antibody, and not the actual quenching/blocknig from my IHC protocol, but I thought I'd check with you guys. Patrick. Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Endogenous Peroxide staining help
Hi everyone, I seem to have a lot of endogenous Peroxide background staining in my FFPE IHC. Human tonsil tissues, with some attached muscle. I do a H202 block at 0.3% H202 in TBS pH 8.0 for 30 minutes. Then I wash x3 with TBST 0.05% Tween20, pH 8.0 Then I serum block with 2% NGS in TBST for 30 minutes. Then I wash x3 TBST Then I add my primary antibody in 2% NGS in TBST overnight. Then I wash x3 TBST Then I add my HRP-labeled 2ndary antibody for 30 minutes. Then I wash x3 TBST Then I have add my AEC Substrate. Should I increase the concentration of H202? Should I increase the time in H202? Is there a different step in the IHC protocol that is better for blocking endogenous H202 activity? Is it possible to lose/reverse the blocking for some reason? I am concerned that my HRP labeled secondary may be to blame. Thoughts? Patrick. Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] More on H202 issues
Hi Everyone Thanks for your responses. I am looking at cell surface markers, Sorry I should have said. Based on what I found out below: Methanol is out, even though I agree that Methanol does enhance the effect of H202 blocking. (I suppose I could try it to see how much/if any epitope loss there is in relation to H202 quenching, At least It would help identifying false positives that are actually H202 artifacts.) Also it looks like increasing the concentration of H202 is out. As to when though, It looks like with cell surface markers I should block after the primary, or even after the 2ndary? Can I do that successfully with a HRP labeled 2ndary? thoughts? Patrick. What solutions or reagents should I use to dilute hydrogen peroxide Methanol, PBS, distilled water or saline can be used to dilute hydrogen peroxide. Morphology of blood smears and peroxidase-rich tissues could be damaged by the aqueous hydrogen peroxide solution. Therefore, methanol is a better choice in this case. Some cell surface markers are very sensitive to methanol/hydrogen peroxide quenching, reducing the staining of antigenic site, particularly on frozen sections. So using hydrogen peroxide in PBS is recommended for cell surface or membrane markers. What concentration of hydrogen peroxide is commonly used 3% hydrogen peroxide is commonly used to block endogenous peroxidase activity. However, certain tissues/cells/antigen (i.e. cell surface markers such as CD4) can be destroyed by high concentration of hydrogen peroxide. So a lower concentration (0.3%) should be used. Where should I do hydrogen peroxide blocking during IHC procedure The blocking can be done (1) after rehydration to water and before antigen retrieval, (2) after antigen retrieval and before primary antibody incubation, (3) after primary antibody incubation, or (4) after biotinylated secondary antibody incubation. For certain antigens such as CD4 and CD8, hydrogen peroxide blocking has detrimental effect on the epitopes, thus reduce intensity of antibody staining. Therefore, blocking after primary antibody or secondary antibody incubation is recommended. Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Can Ice Crystal artifact pick up AEC substrate.
Hi Everyone, I am seeing some red granular staining with my AEC substrate, and I am not sure if its real, or Ice crystal artifact, or debris from the slide directly. It's in several areas and is consistent between duplicates. So can I rule out slide debris? How deep would Ice crystals go in a block. If I cut 10-15 7 uM sections deeper would I eliminate ice crystals from one slide to another? I don't have any pictures yet, but I can try and post it when I do. Patrick. Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Still having Issues with Acetone fixation.
Hi everyone, Sorry to keep posting about this, But I am still having Acetone issues. I am doing IHC for Cell surface markers that are lost when fixing with etoh or methanol. When I fix in 100% acetone my epitopes have great signal. However, when I fix in 100% acetone, my tissues all damaged by the acetone beyond all recognition. I can lose up to 90% of my tissue when I fix in 100% acetone for 10 minutes. But, I get good epitope staining if I have some tissue left on the slide. When I fix in anything else, I lose 90% or more of my epitope staining, but my tissue morphology looks great. -- What's the least amount of time I can fix in 100% Acetone for a 5uM section and still have it be fixed? Is drying after the 100% acetone fixing essential? or Bad for protecting tissue morphology? -- I am doing IHC on human tonsils cut to 5 uM with a 24 dry after sectioning. When I fix in 100% acetone, I fix it at 4C for 10 minutes. Then dry for 1 hour in the fume hood Should it go straight into buffer? Should it be for less time in the acetone? Should the acetone be Room temp or -20C instead of 4C. If I was diluting the acetone with buffer, (or etoh) then I could see going straight into buffer afterwards, but because I am using 100% I think that going into liquid right after fixing is too much of a change and my tissues go BOOM. Please help. Patrick Lewis Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Acetone fixation problems with OCT Tissues
Hi Everyone, I am still having issues with my IHCs with Acetone fixation. If I fix in 100% Acetone, I get IHC staining, but my tissues are 50-90% destroyed. If I fix in 4% paraformaldehyde, or 10% NBF or (95% Etoh and/or Methanol with Acetone) I lose the epitopes I either get no staining or very weak staining, but the tissue morphology look fine. I just tried an acetone gradient where I cut the tissues at 5 uM and dried them overnight, then fixed for 10 minutes in 100% acetone, then fixed in 95% acetone for 1 minute, then fixed in 70% acetone for 30 seconds, then quick rinsed in H20, then washed as normal in DPBS pH 7.4. I did 4 slides, 2 slides with one company's Charged slides ,and 2 slides with another company's charged slides. One company's slides look completely destroyed, the others may turn out, it was hard to tell how much damage there was. I'll know tomorrow when I finish staining and Hemotoxylin them. Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Acetone fixing and tissue damage
Hi Everyone. When I fix my cryosections in acetone, I am using HPLC grade 99.9% for 10 minutes at -20C. Would the Histology grade 99.5% be less damaging to them? Higher H20 content, i.e. less than 99.5% apparently is also very bad. With the HPLC grade I often get tissue damage, the tissue also floats off the slide causing a stringy effect. Fixing with 4% p-formaldehyde or 100% Methanol, prevented the antibody from recognizing the Nuclear Antigens. Looking for advice, Patrick. Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Cryostat purchasing
Hi Everyone, Can anyone recommend a good Cryostat to buy for use with Human Tissues. I'd like to look for a reasonably priced model. Also, I'd consider paying extra for a model that is designed for human tissues so it has disinfection ability or is easier to disinfect than older models. I'd also consider going with a refurbished model if the price was right. I am used to cutting on CM3000 and CM3050s which are quite old. Thanks Patrick Lewis Research Associate II Bench Seattle Childrens Research Institute 206-884-1115 CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Cryostat maintenance personnel
Hi everyone, Does anyone know who I would contact about servicing an old CM3000 Cryostat. This would be in the Seattle area and we have no service contract. I am going to call Leica tomorrow, but if anyone has information about repair/servicing that would be helpful. Thanks Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Cryostat troublshooting. CM3050
H everyone, We have been using this old CM3050 cryostat for our Frozen OCT blocks. I am cutting at 5 uM. My issue is that when I use the hand rotator to cut my sections it doesn't seem to advance with each rotation. It can take as many as 4 rotations to go from one section to another. What would cause this, and is there an easy solution to this problem? I can use the button to automatically move the chick holder all the forward and back from blinking to blinking, so I am wondering if ice crystals or debris can be ruled out as the issue. Any advice welcome. Thanks Patrick. ps: when I do get the blade to cut into the block, I have to wonder if the section I am getting is really 5 uM, or is it thicker. Sometimes you can tell if the section looks particularly thick, but other times it's hard to tell. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Paraformaldehyde fixation questions
After having a lot of problems using 100% Acetone as a fixative, I am going to try 4% paraformaldehyde in DPBS pH 7.4 for fixation of slides cut from fresh tissues snap frozen in OCT. My plan is to cut the slides at 5 or 6 uM and dry them for 2 hours in the hood, then fix them in the 4% p-f for 10 minutes. Then immediately immerse them in Room temp DPBS. Is 10 minutes fixation long enough? Would 30 minutes work better? Is there any advantage, or disadvantage of using 4C versus Room temp for Paraformaldehyde? I am concerned about the drying of the tissues, and tissue loss from the slide. I'm not sure which will be best, drying after cutting or drying after fixing. Years ago, I vaguely remember being told that you should never dry the tissue after fixing it and then re-wet it. So I am leaning towards drying after cutting, but keeping the slides wet after fixing. The goal is for me to get good fixation, not destroy epitopes, and avoid the tissue damage that I get when I fix with Acetone. Thanks Patrick. PS: My brief internet search stated that Fixation occurs faster at higher temperatures (which I knew) , and that Some antibodies are capricious so for them 4C is better. (= IHC myth that corresponds to find what works and then don't change it) Is it right to assume that 4C is going to be gentler on the tissues than Room temp. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] OCT Tissue fall out and reembedding
Hi everyone. If you have several OCT blocks in which the Tissue has fallen out and to want to salvage them for IHC. What is the best way to do so to minimize freeze-thaw damage. These samples were harvested and then were probably snap frozen in Isopentane surrounded by dry ice and stored at -80 when they were made. The people using these blocks now have had several of the blocks in which the tissue has popped out of the block. We are going to get these blocks for IHC. What is the best way to salvage these tissues so that I can do IHC on them. Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Does anyone know where I can get a metal container to hold isopentane that I would place my OCT molds into when making Frozen blocks?
Hi Everyone. I make my Frozen blocks by Immersing the cryo molds in a metal container (Looks like a larger flat base ½ coke can) filled with Isopentane solution. That container is then surrounded by Dry Ice. We need a new metal container. Does anyone know where I could buy one? I don't really want to change to an Aluminum block method as I like the penetration freezing I get from liquid emersion. Thanks Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] OCT Issues
Hi everyone. With Snap frozen tissue in OCT. If I cut sections and fix them in cold acetone for 10 minutes, (Is 2 minutes vs 10 minutes going to cause any issues?) Is it better to dry them and then put them in DPBS or just put them straight into DPBS? If I changed the fixative to 10% NBF would that be better for the tissues than Cold Acetone? I am still having problems with my Frozen sections looking digested, and I am not really sure what is causing it. Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Is there a way to post pictures of slides on histonet? Or to make them available for people to look at?
Hi everyone, What is a good way to post pictures of slides for you guys to look at? I had a weird black precipitate in one of my IHCs and I'd like for you guys to look at them and see if you recognize what this artifact is? Thanks Patrick. PS: I tried uploading them as attachments but the size was too large even though I tried to crop things to keep the size to a minimum. Thanks Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] I just posted 2 photos to histosearch
Thanks everyone, I just posted 2 images to histo search. IS there a way to post up to 5 pics at once? It looked like it suggested that you could post up to 5 pics as a group but I didn't see HOW to do it. Anyway, I have one post of outside of the tissue area, and one post of the tissue area. What is this weird black precipitate? I was using Red AEC as a substrate. Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Wierd hemotoxylin result on my macaque liver slide
Hi everyone. I was staining a FFPE macaque liver tissue slide with hematoxylin the other day, and it had an odd appearance. All the cells stained the usual way with the blue nuclear staining that you expect with hematoxylin. But a few randomly scattered individual cells throughout the tissue stained a sharp nuclear dark black. The cells are not clustered in any way, the cells have sharp black nuclear staining. It's definitely not artifact. Its not a cluster of lymphoid cells like you would see in inflammation. What would cause seemingly random individual cells scattered throughout the tissue to stain black with just hematoxylin? Just curious Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Replacment parts for a Stovall Belly dancer bench top shaker.
Hi everyone, Does anyone know where I can get replacement parts for a Stovall Belly Dancer bench top shaker? One of the 4 tubing legs has snapped near the base. I might be able to jury-rig it with wire and duct tape, but it does have screws at each end of the tubing so It looks like the tubing legs should be replaceable. Thanks Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Best place to Buy Slides boxes preferably True North.
Hi Everyone, Can anyone tell me the cheapest place to get True North Slide boxes (100 slides purple box) VWR has them for about $33.00 EACH. We get a shipping discount with VWR but there has got to be a cheaper supplier. Baring that, can anyone recommend a good 100 slide storage box. Preferably in the $10-20.00 per box range. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Questions about IHC in Frozen Sections
Hi Everyone. I am trying to troubleshoot my IHC on frozen sections. My sections are human tonsil at 7 uM. On charged Superfrost slides. They are stored at -80 after drying for 1 hour. When I use them for IHC, I take them out of the -80 and let them air dry for 1 hour before placing them in cold acetone for 30 minutes to fix. Question: If I place them directly in H20 or TBST pH 8.0 after fixation, will that cause cell lysis? Should I dry the slides after acetone fixation before washing them? If so, for how long? My problem seems to be that the tissue is getting digested on the slide, I am trying to trouble shoot which step is causing my tissues to disintegrate. So far I have tried thicker sections 10, 15 uM (That made the problem worse, I am consider going back to 4 uM sections) I also Changed the concentration of H2O2 for my H202 block from 3% to 0.3%, (In my next IHC attempt I will try to examine the slide at each step to see if I can see loss of integrity) Also in my next attempt I plan to eliminate any H20 washes and dry the slide post acetone fixation before washing in TBST. Also I plan to decrease the amount of Tween20 in my Wash buffer from 0.2% to 0.02%. Any advice would be helpful. Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Has anyone had any problems with the Vector Immuedge pap pen?
Hi everyone, Has anyone had any problems with the Immedge Pap pen from Vector. Cat # H-4000 It gives a nice wide barrier, but for some reason the barrier is disintegrating on our slides so I get holes in the lines I draw. I have had a similar problem before, usually because the slide wasn't dry enough when I was drawing the barrier or I was immersing the slide before the barrier had a chance to solidify. However, the problem has never been this bad. I am wondering if my current batch of pap pens is defective. Patrick CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Can someone recommend a good labeling pen for cryotubes.
Hi everyone, Can someone recommend a good pen to use to label cryo tubes.The VWR pens we have been using are apparently discontinued. I would like something that writes as fine as an ultrafine sharpie, but is less likely to smear and is more long lasting, and solvent tolerant. Thanks Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] nanodrop of DNA preps from paraffin sections
Hi Everyone, Just out of curiosity, what sort of nanodrop values do you typically get with DNA extractions from paraffin sections. I usually get around 100 ng/uL, depending on tissue size, and the number of churls I take. Around 80% are 100 ng to 200 ng/uL. Rarely, I get as high as 1000 ng/uL, 0-5% of the samples I take. Occasionally I get 10-40 ng/uL 5-15% of the samples I take. I primarily extract from tonsil, but I have done several other tissue types. Patrick. I also primarily use QIAgen Mini kits. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Searching for HHV-7 antibody
HI everyone, Does anyone know where to get 5E1 mouse monoclonal antibody directed against HHV-7 (pp85 protein complex) We tried two suppliers but their product has been discontinued and now we can not seem to find it anywhere. Thanks Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Can someone recommend a good means of storage for Cryomold blocks?
Hi everyone, can someone recommend a good storage method for Frozen tissue blocks made from cryomolds? We have about 200 blocks. Currently we have them stored freezer boxes in bags with some wet ice at -70. It might be better if we had plastic tray bins to put the bags in, but I have not been able to find them yet. Anyone have a catalog number for those gray tray bins? Thanks Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Can anyone recommend a good Rabbit Isotype Control antibody
Hi Everyone, Can anyone recommend a good Rabbit Isotype Control Antibody IgG1, to use as a negative control for my Polyclonal Rabbit antibodies. I am looking at Biocompare and GenTex has one for about $220.00 for 100 ug. GTX47478 But if someone has one they've used and works well, I'd be interested. Thanks Patrick, ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Has anyone done ISH with fairly large FFPE sections?
Hi Everyone, I am going to do some ISH for EBER on FFPE sections, and I have some concerns on the logistics. The kits I am looking at are for 50 rxns, and it seems like they expect to use 20 uL probe per rxn. My sections are rather large, covering about 2/3 of a standard slide. How many uL will it take to make sure I get good coverage and have enough to avoid drying out the slide? I plan to cover slip them and use a humidity chamber, but even still, I am worried that with such a small volume it don't be enough. These kits are expensive, +$900.00 each and if I have to double/triple my rxn volume that would use up the kit in a hurry. I haven't fully familiarized myself with the protocol yet, so maybe my concerns are unjustified, but if anyone has done some ISH, I'd appreciate any advice you'd care to give. Thanks Patrick. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Preferences between kits for EBER
Hi Everyone, Thanks for the response earlier. Does anyone any advice on EBER ISH Kits? Specifically, if they had better results, or less problems with one brand vs another? Right now I am considering both Novacastra's kit for 970.00 and Vector Labs kit for 905.00 Has anyone used these kits? Has anyone tried a different kit they could recommend? Thanks Patrick. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Antibodies to Mast cells for IHC help
Hi everyone. Would anyone be able anyone recommend a good antibody to stain mast cells in Human Tonsil for IHC? These would be in FFPE (Formalin fixed paraffin embedded sections) With secondary antibody hopefully being HRP labeled. Substrate would most likely be DAKO AEC. Thanks Patrick Lewis PS: If you have done this staining in the past and have a protocol, or antigen retrieval suggestions that would also be helpful. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Antibodies to mast cells in human tonsil
Hi everyone, That should read, Would anyone able to recommend a good antibody to stain for only mast cells in human tonsil for IHC. Thanks in advance. Patrick Lewis CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] H2O2 Recep
So what I'm gathering from all this. Thanks by the way for the discussion. Methanol is unnecessary for the H2O2, reaction, but it can make it slightly faster since both methods will be working in concert. If you have tissue that has more peroxidase than usual, then extra time is required. Also, If you use straight aq H2O2 on frozen sections the bubbling may damage the tissue. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Methanol in H2O2 explanation
Hi everyone, Can someone refresh/enlighten me as to why Methanol is used in H2O2 blocking. Let's assume that I take my slides through the xylene/ethanol steps and that after the 70% etoh wash step, they are now in TBST. I could see adding methanol if you were doing the H2O2 blocking as part of the xylene/etoh process and maybe you had the methanol as a substitute for the 70% etoh. My xylene/ETOH looks like this. 3 x 5 min xylene. 2 x 5 min 100% etoh 2 x 5 min 95% etoh 1 x 5 min 70% Then my slides are washed and then epitope retrieved. After epitope retrieval they are again washed and then I do my H2O2 block without methanol. Thanks Patrick. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Recommendations for HRP anti Rabbit and mouse and AEC substrates
Hi everyone, I have been using DAKO Kits for HRP anti rabbit and HRP anti Mouse and AEC substrates. I love DAKO, but their kits and reagents are expensive. Can anyone offer an alternative for HRP labeled anti (Rabbit/Mouse) antibodies and AEC substrate kits. I do all my IHC manually by hand. I am doing my IHCs primarily on FFPE slides of various tissues, some of which are large enough that they take up a lot of the slide, and hence need more drops to get complete coverage. Thanks Patrick. PS: I am considering Vector Labs, and I am also looking at Biocompare to seek out more vendors. Has anyone tried Spring Bioscience's products? CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Coverslip removal question
Hi Histonetters When I soak my slides in xylene to remove the coverslip, do I then need to rehydrate them by washing them in 100% etoh,95% etoh,70% etoh, and then H20. And then drying and re applying permount? Or can I go straight from xylene to drying to re-coversliping with permount? Patrick Lewis Research Associate II-Bench| Infections and Prematurity Seattle Children's Research Institute 206-884-1115 OFFICE 000-000- PAGER 000-000- CELL 206-884-7311 FAX patrick.le...@seattlechildrens.org OFFICE 1900 9th Avenue Seattle, WA 98101 MAIL M/S C9S-8, Seattle, WA 98101 WWW seattlechildrens.org http://seattlechildrens.org/ CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Recommendations for decal solution
Hi guys, Can anyone recommend a good decal solution. I have some bone marrow and trachea tissues for paraffin sectioning and I want to decal them. Thanks Patrick Patrick Lewis Research Associate II-Bench| Infections and Prematurity Seattle Children's Research Institute 206-884-1115 OFFICE 000-000- PAGER 000-000- CELL 206-884-7311 FAX patrick.le...@seattlechildrens.org OFFICE 1900 9th Avenue Seattle, WA 98101 MAIL M/S C9S-8, Seattle, WA 98101 WWW seattlechildrens.org http://seattlechildrens.org/ CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] decal solution help
Dear Histonetters Yes I should have said, I'll be doing IHC for viral proteins with both polyclonal (rabbit) and monoclonal (Rat/Mouse) primary antibodies and HRP-labeled secondary antibodies with DAKO AEC Substrate. Patrick Lewis Research Associate II-Bench| Infections and Prematurity Seattle Children's Research Institute 206-884-1115 OFFICE 000-000- PAGER 000-000- CELL 206-884-7311 FAX patrick.le...@seattlechildrens.org OFFICE 1900 9th Avenue Seattle, WA 98101 MAIL M/S C9S-8, Seattle, WA 98101 WWW seattlechildrens.org http://seattlechildrens.org/ CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] I have a couple of quick questions
If I want to remove a cover slip that is coversliped with permount. Do I need to use xylene to get rid of the semi hardened permount gunk? Also, If I am heating paraffin sectioned slides overnight at 37C to adhere them what happens if I over cook them by leaving them at 37C for more than one day? Say 3-4 days at 37C It probably wont hurt them as they haven't been epitope retrieved yet. Patrick Lewis Research Associate II-Bench| Infections and Prematurity Seattle Children's Research Institute 206-884-1115 OFFICE 000-000- PAGER 000-000- CELL 206-884-7311 FAX patrick.le...@seattlechildrens.org OFFICE 1900 9th Avenue Seattle, WA 98101 MAIL M/S C9S-8, Seattle, WA 98101 WWW seattlechildrens.org http://seattlechildrens.org/ CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Sucrose cryoprotection
Hi Histoneters Can someone explain sucrose cryoprotection to me. Why/when is it necessary? Tissue types that require it? Potential benefits/problems with its use or not use? Thanks. Patrick Patrick Lewis Research Associate II-Bench| Infections and Prematurity Seattle Children's Research Institute 206-884-1115 OFFICE 000-000- PAGER 000-000- CELL 206-884-7311 FAX patrick.le...@seattlechildrens.org OFFICE 1900 9th Avenue Seattle, WA 98101 MAIL M/S C9S-8, Seattle, WA 98101 WWW seattlechildrens.org http://seattlechildrens.org/ CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] NBF fixation and long term storage
I have some questions about Fixing tissues for paraffin embedding when the ultimate purpose will be looking for viral antigens and cell surface markers. We have some monkey tissue that has been in NBF for over a year. and we are just now processing them and embedding them in to paraffin. We will be getting additional tissues from monkey necropsies and in an effort to avoid epitope damage from prolonged exposure to NBF I was wondering if anyone could recommend a fixation period. (I.E. no longer than 24 hours in NBF, 48 hours? ETC. I was thinking of replacing the NBF (Neutral buffered Formalin) with 70% Etoh. And using 70% Etoh as long term storage of tissue parts. The ideal situation would be to get the tissues from necropsy the same day of the necropsy and store the tissues overnight in NBF and then transfer them to 70% etoh and then process them on the tissue processer after 24 hours in Etoh. I'm not sure how many animals I've eventually get and I'm a little concerned over where we will put all the spare animal parts that I'm sure I'll accumulate. We don't want to throw anything away and for some tissues I'll have more than I can put into a single cassette. I may end up using more than 1 cassette for a single piece of tissue so that I have backup cassettes of it. Anyone storing research tissues has a suggestion about the best way to store them when space is a premium. Thanks Patrick. Patrick Lewis Research Associate II-Bench| Infections and Prematurity Seattle Children's Research Institute 206-884-1115 OFFICE 000-000- PAGER 000-000- CELL 206-884-7311 FAX patrick.le...@seattlechildrens.org OFFICE 1900 9th Avenue Seattle, WA 98101 MAIL M/S C9S-8, Seattle, WA 98101 WWW seattlechildrens.org http://seattlechildrens.org/ CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Quick frozen sectioning and IMHC questions
Hi everyone, I cut my first frozen sections, human tonsil, and I have stored them at -70. When I take them out to use, should I bring them to -20C and fix them right away? Or should I bring them to room temp and then pap pen/wash/block/wash them then fix them before adding my primary anti-body. My original plan was/is to bring them to room temp, pap pen them and then wash/block/wash fix. I am asking for your advice as my boss is away for two weeks and the protocol he left me with is a bit vague and requires some modifications to fit the research that we are doing. I was just wondering if there is a compelling reason to keep the sections at/near -20 after sectioning up through the fixation before starting the Immunohistochemistry. Also, we are using BSA as one of the components in our blocking solution as well as a component in our wash solution. Is there a danger in using lower/higher quality BSA? I'm a little worried that our BSA might contribute to increased background if its quality is not high enough. The BSA we may use is A-8327 Sigma, and I am thinking this might be better A3294, or one of these (A9647,A7906,A6793) This is a new job for me, and its been a long while since I have done any Immunohistochemistries.so I am going over the reagents we have on hand while I refresh my memory on immunohistochemistry and sectioning techniques. Thanks for all your support Patrick Patrick Lewis Research Associate II-Bench| Infections and Prematurity Seattle Children's Research Institute 206-884-1115 OFFICE 000-000- PAGER 000-000- CELL 206-884-7311 FAX patrick.le...@seattlechildrens.org OFFICE 1900 9th Avenue Seattle, WA 98101 MAIL M/S C9S-8, Seattle, WA 98101 WWW seattlechildrens.org http://seattlechildrens.org/ CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Another quick sectioning question
When I was cutting my block I noticed some ribboning of the tissue. Is there a known cause (or several) for this. I am wondering if my microtome blade was too tight or too loose or if my block froze poorly, or if I just needed to reorient my block . I was cutting 7 um at -20. Its human tonsil, It's been a long time since I used a cryostat (we're using a leicaCM3000).The ribboning almost disappeared when I changed blades after cutting about 30 sections into the block. But I still had some ribboning of the tissue even though I was getting pretty clean OCT slices away from the tissue. Also, anyone have advice on disinfecting cryostats. I will eventually be cutting infectious tissues, HIV human samples and NHP macaques. We plan to follow Leicas disinfection procedure in which you bring the cryostat to Room temp then spray with 5% Bleach let it sit for 10-20 minutes then wipe and spray with 70% ethanol and sit for 10 min then wipe and spray with 100% ethanol. And drain and wipe. Anyone have advice on cleaning and if it's necessary to remove any portions of the cryostat to facilitate cleaning. I figure it will be a two to three day process. To bring it to room temp, clean it, then bring it back down to -20. My main concerns are making sure its disinfected, and then making sure we remove all the bleach so that we don't get corrosion. Thanks for all your help and advice. Patrick. Patrick Lewis Research Associate II-Bench| Infections and Prematurity Seattle Children's Research Institute 206-884-1115 OFFICE 000-000- PAGER 000-000- CELL 206-884-7311 FAX patrick.le...@seattlechildrens.org OFFICE 1900 9th Avenue Seattle, WA 98101 MAIL M/S C9S-8, Seattle, WA 98101 WWW seattlechildrens.org http://seattlechildrens.org/ CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Greetings and IMHC Questions
Hello everyone, My name is Patrick Lewis and I just started working for Children's Hospital in their Research Department. It's been a long time since I've done any Immunohistochemistry, (~10 years), but in this new job, I will be doing a lot of it with both paraffin and frozen sections that I will make myself. Anyway, I am about to try staining my first frozen slides and I have a couple of quick questions. I cut some practice slides, human tonsil, and have stored them at -80 in a slide box. How much longevity will they have? I'd like to be able to cut slides and store them unfixed at -80 for up to one month until I use them I know its better to use them the same day, but it would be nice to be able to cut them on one day and use them as needed Also, is there a good place to find general IMHC procedures? It's been a long time and I want to re-familiarize myself with some general protocols I am using Superfrost Plus slides, but we may have some problems with the sections falling off the slide. Does anyone have any suggestions on methods to avoid tissue loss from the slide? Also, I am freezing my tissues in OCT in cryomolds that are lowered into an iso-pentane filled ½ coke can which is immersed in a bucket of dry-ice for 5 minutes. . I wanted to avoid using liquid nitrogen and am hoping that with the dry ice method I would still get good freezing of the block. Are there any recommendations for OCT freezing? Thanks for all your help. Patrick. Patrick Lewis Research Associate II-Bench| Infections and Prematurity Seattle Children's Research Institute 206-884-1115 OFFICE 000-000- PAGER 000-000- CELL 206-884-7311 FAX patrick.le...@seattlechildrens.org OFFICE 1900 9th Avenue Seattle, WA 98101 MAIL M/S C9S-8, Seattle, WA 98101 WWW seattlechildrens.org http://seattlechildrens.org/ CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information protected by law. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
