[Histonet] Glycogen detection
Dear Colleagues,I really appreciate your prompt and detailed responses to my question about glycogen detection. Thank you for jolting my memory, I think I'm starting to forget the biochemistry basics. I'm gonna review the histochemical textbooks. Your advice is very helpful and I will definitely follow the suggestions. Tank you again! Sincerely, Galina Deyneko ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] Glycogen detection
Dear Colleagues.Does anybody have experience how fix the tissues for successful glycogen detection in murine and humane cardiomyocytes.I am wondering maybe the trace of methanol in 10% formalin will dissolve glycogen? What would be better process for paraffin embedding or use OCT embedding without fixation. Of course I prefer FFPE blocks, since OCT blocks give bad morphology.Any advices?Great thank you in advance Galina Deyneko ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] picro sirius red staining
Please see my protocol, we successfully use it on murine liver and hearts.I do not stain nuclei, in my opinion counterstaining with any type of hematoxylin contaminates and fades picris acid. - Sirius Red F3BA(Polysciences #09400), (Direct Red 80 from Sigma Chem. Co. (Cat# D 030)) - Saturated aqueouspicric acid solution (LabChem Inc. #LC 18670-2) - Picric acidpowder (Spectrum #P1145) - Phosphomolybdicacid (Sigma #HT153) - Hydrochloric acid(Sigma #H1758) - Bouin’solution 0.1% solution of Sirius Red: 0.2 gSirius Red powder 200 mlsaturated aqueous solution of picric acid - Add alittle amount of solid picric acid to ensure saturation. Stir well, filterbefore use. - Stablefor 1 year and can be used many times. 02% solution of phosphomolibdic acid: 4 ml of10% phosphomolibdic acid 200 ml DIwater 0.01N solution of hydrochloric acid: 166 µl of38% HCL /830 µl 200 ml DIwater /1 L Procedure: ·De-waxSlides ·Incubatein 0.2% solution of phosphomolibdic acid (optional) ·Mordantovernight in saturated solution of picric acid at room T ·Enhancethe yellow color by mordant in saturated solution of picric acid additional 1- 2hour at 60 C in the oven. ·Stain in0.1% solution of Sirius Red for 2 hours at RT ·Rinse in onechanges of 0.001 N solution HCl- very quick dip!! ·Dehydratein four changes of fresh!! 100% Ethanol also quick deep in each ·Clearslide through three changes of xylene ·Coverslipusing a permanent mounting media Results: The PSRed method stains mostly collagen types I, II, and III. Light microscopy: collagenous fibers – red, other tissue elements - bright yellowPolarized microscopy: collagen fibers - orange/red bands against black background Galina Deyneko Novartis, CVM galinadeyn...@yahoo.com ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] XGal staining
Dear All, and Anna.I am confused about the recommendation of post fixation (stained slides?) in glutaraldehyde-foormaldehyde solution. According many protocols and my experience the fresh tissues should be fixed in in glutaraldehyde -formalin solution that is mproved the tissue morphology and staining quality.The best counterstaining is Nuclear Fast Red from Vector. Galina DeynekoNovartis, Cambridge, MA 617-871-7613 w ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] pre-floating tissue sections
Hi Colleagues, I also use this technique with intermediate bath with cold DI water and like it. also i breath on the surface of the block befor make one section, this helps making the section more smooth. Galina DeynekoNovartis, Cambridge, MA 617-871-7613 w From: histonet-requ...@lists.utsouthwestern.edu histonet-requ...@lists.utsouthwestern.edu To: histonet@lists.utsouthwestern.edu Sent: Sunday, June 7, 2015 1:00 PM Subject: Histonet Digest, Vol 139, Issue 7 Send Histonet mailing list submissions to histonet@lists.utsouthwestern.edu To subscribe or unsubscribe via the World Wide Web, visit http://lists.utsouthwestern.edu/mailman/listinfo/histonet or, via email, send a message with subject or body 'help' to histonet-requ...@lists.utsouthwestern.edu You can reach the person managing the list at histonet-ow...@lists.utsouthwestern.edu When replying, please edit your Subject line so it is more specific than Re: Contents of Histonet digest... Today's Topics: 1. Re: Paraffin block disposal (Aimee Tolentino) 2. Re: Paraffin block disposal (Cooper, Brian) 3. Re: tissue fixation-formaldehyde concentrations (Hobbs, Carl) 4. Re: Paraffin block disposal (Brendal Finlay) 5. Re: Pre-floating tissue sections in dilute alcohol (tjfinney2...@gmail.com) 6. Re: Decalcification of bone marrows (Bob Richmond) 7. Re: Pre-floating tissue sections in dilute alcohol (Joana Moreira) 8. Re: Pre-floating tissue sections in dilute alcohol (Gudrun Lang) -- Message: 1 Date: Sat, 6 Jun 2015 10:24:42 -0700 From: Aimee Tolentino a.tolentin...@gmail.com To: Arbaugh, Roberta rarba...@csdermatology.com Cc: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Paraffin block disposal Message-ID: 1f36aba5-452b-4556-8d1e-e5d09fdb2...@gmail.com Content-Type: text/plain; charset=us-ascii That's a good question. I'd like to know the answer myself to that. :) Sent from my iPhone On Jun 5, 2015, at 12:54 PM, Arbaugh, Roberta rarba...@csdermatology.com wrote: Per CLIA we only need to keep paraffin blocks two years. What is the proper way to dispose of them? DISCLAIMER: The information in this message is confidential and may be legally privileged. It is intended solely for the addressee. Access to this message by anyone else is unauthorized. If you are not the intended recipient, any disclosure, copying, or distribution of the message, or any action or omission taken by you in reliance on it, is prohibited and may be unlawful. Please immediately contact the sender if you have received this message in error. Thank you. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Message: 2 Date: Sat, 6 Jun 2015 18:34:17 + From: Cooper, Brian bcoo...@chla.usc.edu To: a.tolentin...@gmail.com a.tolentin...@gmail.com Cc: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Paraffin block disposal Message-ID: c12647ad4408834c8ab48fb0389c63e3e4243...@chlaexmbh01.la.ad.chla.org Content-Type: text/plain; charset=us-ascii Hey Aimee, This has been discussed several times on Histonet. It sounds like it depends on the institution. Since they're FFPE, pathogens are not a concern. I didn't reply to all because someone will shout out, What about CJD? and then I would have to punch them. They should be able to go into the regular trash though, since there is nothing that anyone can catch from them. Here, just like Genzyme, we are told to dispose of them as regulated, biohazard waste. You would have PHI concerns if the patient's name is on them, so they'll need to be identified first . . . Thanks, Brian Cooper, HT (ASCP) Supervisor, Histology Children's Hospital, Los Angeles Sent from my Galaxy S5, so please forgive any weird typos . . . -Original Message- From: Aimee Tolentino [a.tolentin...@gmail.com] Received: Saturday, 06 Jun 2015, 10:25AM To: Arbaugh, Roberta [rarba...@csdermatology.com] CC: histonet@lists.utsouthwestern.edu [histonet@lists.utsouthwestern.edu] Subject: Re: [Histonet] Paraffin block disposal That's a good question. I'd like to know the answer myself to that. :) Sent from my iPhone On Jun 5, 2015, at 12:54 PM, Arbaugh, Roberta rarba...@csdermatology.com wrote: Per CLIA we only need to keep paraffin blocks two years. What is the proper way to dispose of them? DISCLAIMER: The information in this message is confidential and may be legally privileged. It is intended solely for the addressee. Access to this message by anyone else is unauthorized. If you are not the intended recipient, any disclosure, copying, or distribution of the message, or any action or omission taken by you in reliance on it, is prohibited and may be
[Histonet] Re: gelatin
Thank you Dr. Kiernan, Nice and scientific explanation how to prepare gelatin. I beleive that a lot of house wives and cooks know how to prepare meet or fish jelly (long bouling of the bones with cartilage and tendons). very popular in russian or easter europian cuisine, called cholodez which neans cold.best regards Galina DeynekoNovartis, Cambridge, MA 617-871-7613 w From: histonet-requ...@lists.utsouthwestern.edu histonet-requ...@lists.utsouthwestern.edu To: histonet@lists.utsouthwestern.edu Sent: Wednesday, December 31, 1969 7:00 PM Subject: Histonet Digest, Vol 135, Issue 25 Send Histonet mailing list submissions to histonet@lists.utsouthwestern.edu To subscribe or unsubscribe via the World Wide Web, visit http://lists.utsouthwestern.edu/mailman/listinfo/histonet or, via email, send a message with subject or body 'help' to histonet-requ...@lists.utsouthwestern.edu You can reach the person managing the list at histonet-ow...@lists.utsouthwestern.edu When replying, please edit your Subject line so it is more specific than Re: Contents of Histonet digest... Today's Topics: 1. Re: Storing antibodies in a frostless freezer (Teri Johnson) 2. Re: gelatin (John Kiernan) 3. RE: [EXTERNAL] Re: [Histonet] gelatin (Roy, Ryan) 4. How dark is dark enough? (Paula Sicurello) 5. Re: gelatin (Yak-Nam Wang) 6. Re: How dark is dark enough? (Rene J Buesa) 7. Creutzfeldt-Jakob Disease (Scott, Allison D) 8. RE: Creutzfeldt-Jakob Disease (Debra Siena) 9. RE: How dark is dark enough? (Morken, Timothy) 10. CBG recycler (Roy, Ryan) -- Message: 1 Date: Mon, 23 Feb 2015 18:28:54 + From: Teri Johnson tejohn...@genoptix.com Subject: [Histonet] Re: Storing antibodies in a frostless freezer To: histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: f1d2836e336c4479936999f03ac57...@phuscb-sp37mb04.genoptix.org Content-Type: text/plain; charset=WINDOWS-1252 I agree with Rachel. I would not be quite as worried about the temperature change if indeed they maintain no higher than -13 degrees C and would not be freeze/thawing, but what does your ice cube tray look like after about a month or more in a frost-free freezer? Best wishes, Teri Johnson, HT(ASCP)QIHC Genoptix, Inc. A Novartis Company Carlsbad, CA CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and contains information that is confidential and proprietary to Genoptix Medical Laboratory or its subsidiaries. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, immediately contact the sender by e-mail and destroy all copies of the original message. -- Message: 2 Date: Tue, 24 Feb 2015 00:47:50 -0500 From: John Kiernan jkier...@uwo.ca Subject: Re: [Histonet] gelatin To: Yak-Nam Wang ynw...@u.washington.edu, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: 7390897614c8f.54ebc...@uwo.ca Content-Type: text/plain; CHARSET=US-ASCII You need to explain treated tissue. Gelatin is collagen that has been boiled until the protein has lost all its fibrous nature and changed into a water-soluble protein. Gelatin is made permanently insoluble by adequate formaldehyde fixation. It is stained by anionic dyes (such as eosin in the HE method), but it does not show as fibres when you look at the section or smear through a microscope. If this doesn't answer your question, please explain your problem and involve your boss in future email exchanges. John Kiernan London, Canada = = = On 23/02/15, Yak-Nam Wang ynw...@u.washington.edu wrote: Hello, Does anyone know of a stain specific for gelatin? I would like to distinguish between firbous collagen and gelatin in treated tissue. thank you Yak-Nam University of Washington Seattle, WA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet -- Message: 3 Date: Tue, 24 Feb 2015 10:06:11 -0500 From: Roy, Ryan ryan@va.gov Subject: RE: [EXTERNAL] Re: [Histonet] gelatin To: 'John Kiernan' jkier...@uwo.ca, Yak-Nam Wang ynw...@u.washington.edu, histonet@lists.utsouthwestern.edu histonet@lists.utsouthwestern.edu Message-ID: 15f883394eab744e99e1c7e1b98730490178a821d...@r04bynmsgb1.r04.med.va.gov Content-Type: text/plain; charset=us-ascii That's interesting, I didn't realize gelatin is the water soluable protein of collagen. No experience with staining gelatin, but have you considered MassonTrichrome. It is used to differentiate collagen from smooth muscle... -Original Message- From: histonet-boun...@lists.utsouthwestern.edu
[Histonet] Re: mouse carotid body
Dear Colleagues. Please share your expertise how to reveal the carotid body in mouse carotid artery. I received from researcher the piece of mouse carotid artery with bifurcation on internal and external carotid branches. What is the best way for embedding: flat parallel to the mold bottom or on transverse axis, that is perpendicular to the mold bottom.? I think I should collect many serial sections and maybe in couple of them I will be able to find carotid body. Am I right? Please advise better and rational way to do this.Thank you Galina DeynekoNovartis, Cambridge, MA 617-871-7613 w ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Sections contamination after Lac Z staining
Dear Colleagues I search your advices regarding Lac Z staining on frozen OCT embedded brain. After staining in Xgal staining solution overnight at 37C all sections covered by glassy small particles. The sections themselves are not contaminated, i checked them with HE staining and also checked under microscope before placing in the beta gal staining solution. The blue Lac Z staining is good and intense. Short method description:fix brain in freshly prepared Glutaraldehyde-Formaline fixative 2 hours, after Sucrose embed in OCT. Section thickness 7 microns. For staining I use Millipore kit. Of cause I warm the stain base solution at 37C before to dilute melted beta Gal stock and the staining solution is clean and transparent, no precipitation. After staining fix sections in 10% formalin for 15 minutes and counterstain in Nuclear Fast red for 40 seconds. I have try several modifications: no rinse the sections in the DI water, rinse the sections in DI water to get rid of OCT, after staining, and after counterstaining, nothing help. I am even not able to catch the moment when appears this contamination. maybe filter the beta gal staining solution before placing slides? please share your suggestions, or detailed protocols. Thank you in advance Galina Deyneko Novartis, Cambridge, MA 617-871-7613 w ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] BRDu IHC protocol
Hi Luis I attach my protocol for BRDu Best regards BRDU protocol 1. De-wax and hydrate through the changes of Xylenes and graded Ethanols (100%, 95%, 70%) the paraffin slides till DI water. 2. HIER :Performed in Biocare’s Decloaking Chamber with manufacture settings ( set point 1-125˚C, 30 seconds, set point 2- 90˚C , 10 seconds) in plastic container with 1X Rodent Decloaker solution which is stable modified Citrate Buffer pH 6.00, (Biocare # RD913). Optional: Place the slides in preheated till 90- 100 ˚C in 1X Rodent Decloaker solution which is stable modified Citrate Buffer pH 6.00, (Biocare # RD913) and steam 45 minutes in steamer .Remove the slides from steamer and allow them to cool till RT˚ 3. Rinse in DI water and Wash in Tris buffer three times x 5 minutes 4. Circle the tissue on the slides with hydrophobic barrier using PAP pen 5. Incubate with pepsin enzyme digestion solution – 2 minutes. 6. Prepare fresh before using: 0.25% solution of pepsin (250 mg of powder in 100 ml of Tris) (powder, Biomedicals, # 195367 ) in 1X Tris buffer( 10X Fisher Scientific, BP2471), titrated to pH 2.00 with concentrated hydrochloric acid. Optional: Preparation of 0.05M Tris(which correspond to 1X Tris) from 1M Tris (Teknova. ,cat.# T1068,pH6.8)- 10ml of 1 M Tris + 190 ml of DI water. (53 mg of powdered pepsin in 22.200 ml of Tris, pH2.5) 7. Neutralize the slides 2 times x 5 minutes each in the 0,1 M Borate Buffer , pH 8.5 (0.5M sodium Borate Buffer, Boston Bioproducts, # BB-66, 1 part of 0.5M buffer + 4 parts of DI water). 8. Wash in Tris buffer three times x 5 minutes. 9. Quench the endogenous peroxidase with PEROXIDAZED 1 (Biocare # PX 968) for 15 minutes at RT 10. Rinse in DI water and Wash in Tris buffer two times x 5 minutes 11. Block endogenous Protein with Background Sniper for 30 minutes at RT ( Biocare , #BS966) 12. Tap off the excess of protein block , do not rinse 13. Incubate the slides with the primary antibody (mouse monoclonal anti- bromodeoxyuridine, Roche, # 11 170 376001) 80 minutes. Diluent: ready-to-use antibody diluent, (Dako, # S0809). Dilutions: 1:700 Wash in Tris buffer three times x 5 minutes 14. Incubate the slides with mouse –on-mouse HRP Polymer (Biocare, #MM620) for 30 minutes at RT.-for mouse tissues or with mouse on rat HRP polymer (Biocare, # MRT 621) for rat tissues. 15. Wash in Tris buffer two times x 5 minutes 16. Stain the slides with freshly prepared DAB chromogen (Dako, #K3468) and developed end-colored product in the targeted cells under microscope. Staining is complete when the protein positive areas turn to dark brown and protein negative areas (background) remain colorless. Stop the chromogen reaction by placing the slides in DI water with following rinse in fresh DI water to remove the residual DAB and clear the slides.- 5 minutes. Galina Deyneko Novartis, Cambridge, MA 617-871-7613 w___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Treatment of calcified human arteries
Dear Colleagues Please share your expertise on treating heavily calcified human arteries. It is impossible to cut them without deminiralization in my opinion. Should I use decalcification solution before processing? If yes, what reagent do you recommend taking in account that I will do IHC for macrophages and other targets on these samples, as well as Sirius Red and Masson Trichrome staining. Thank you in advance Galina Deyneko Novartis, Cambridge, MA 617-871-7613 w ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: cell pellet preparation
I attach my protocol for cell pellet Preparation of formalin fixed Cell pellets. 1. Grow up approximately 5-1o millions cells. 2. Centrifuge the cells with the medium in conical tubes for 10 minutes with speed 1000rpm. 3. Remove supernatant, wash once with 1X PBS at RT, centrifuge as before and discard PBS supernatant. 4. Place 2 ml or more of 10 % formalin in tube, flick to break up pellet, and re-suspend the cells in formalin and fixed 20 minutes at RT. 5. Centrifuge as before and remove formalin supernatant. 6. Wash once with 1X PBS, at RT, centrifuge as before and discard PBS supernatant. 7. Add 1 ml of 1X PBS, carefully re-suspend the cells and transfer to 1.5 ml Eppendorf tube. 8. Microfuge 10 minutes at 0.8 xg (2900 rpm) and remove as much PBS supernatant as possible. 9. Warm the Histogel (Allan Scientific # HG4000-012) in microwave oven 10-20 seconds till Histogel turns to liquid. Let it cool a bit. 10. Re-suspend cells in 100-150 µl of warm Histogel, leave pellet undisturbed for at least 20 minutes in wet ice for gel solidifying. 11. Remove the solid gel pyramid with sharp wood stick, wrap in histo paper place in cassette and process Galina Deyneko Novartis, Cambridge, MA 617-871-7613 w ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re:Rabbit antibody on rabbit tissues
Dear Colleagues My current problem is: I should detect the myeloperoxidase (MPO) antigen and its derivate nitrotyrosine and chlorotyrosine in the rabbit tissues (aorta) . Most of the antibodies what I found and claimed to work on Rabbit are rabbit polyclonal. Do you know antibodies for those antigens from any other species or biotinylated antibodies, or you can suggest good secondary antibodies and detection systems that might work on rabbit tissues with rabbit primary, on the analogue of mouse-on-mouse detection system. I already tested goat and mouse anti-human MPO on rabbit aorta and lung/liver as a positive control, but besides stained macrophages and endothelial cells, i got stained smooth muscles in the media, and I am not sure that the staining in macrophages is specific. Maybe yes, since negative controls with mouse and goat IgG are absolutely clean. Can you share you advices, opinion, experience. Thank you in advance Galina Deyneko Novartis, Cambridge, MA 617-871-7613 w --- On ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] (no subject)
Hi Betsy We use the Thermo Shandon precision Cut paraffin cat. # B1002490 ( Thermo Scientific) Galina Deyneko Novartis, Cambridge, MA 617-871-7613 w ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Histonet Digest, Vol 106, Issue 35
Dear Colleagues I would like to ask again about cell block preparation. of cause I found number of answer on the histonet site and sorry that I ask again. The cells what I prepare look distorted. Short protocol: 30 minutes of fixation in 10 %NBF, centrifuge 1000 rmr, wash in PBS, centrifuge, re-suspend in histogel and processed in Thermo Shandon with following program: Ethanols70, 70,80, 95,95,100,100,100,Xylenes 3 changes, Wax 3 changes - 1 hour in each station. I also tried short protocol - 30 minutes in each station. I also would like to try to do OCT embedding to avoid processing anparaffinin embedding. Should I fix the cells before OCT embedding or not? Can I re-suspend directly in OCT or still need to embed in Histogel first and freeze in OCT Histogel block. Please could you share your protocols and give me some hints. Thank you and good weekend. Galina Deyneko Novartis, Cambridge, MA 617-871-7613 w ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] macrophage stain
I use rat-a- mouse Mac3 on FFPE mouse aortic sinus. BD pharmigen # 550292,very simple basic protocol Dilution 1;200, no need HIER with tis dilution. Galina Deyneko Novartis, Cambridge, MA 617-871-7613 w ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: IHC in porcine tissues
Dear Colleagues What secondary antibody or polymer would be better to use for mouse primary antibody on FFPE porcine tissues?Also, please share your experience regarding macrophages detection in porcine arteries. I found couple in Abcam and Serotec (only for frozen), but your opinions is very valuable. Thank you Galina Deyneko Novartis, Cambridge, MA 617-782-1675 home 617-871-7613 w ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Histonet Digest, Vol 87, Issue 12
Dear Colleagues, I would like to find chromogen for alkaline phosphatase enzyme labeling. I have used Vulcan fast red from Biocare, but it usually gives me background especially on double stained slides on mouse pancreas, i do KI 67 and BRDU/ Insulin double staining, and I use AP enzyme for the pancreatic islets with rabbit S Cruz anti insulin antibody and rabbit--on rodent AP polymer from Biocare Could you please share your experience what and from what company detection system and chromogen i can use to get rid of background. I appreciate any hints and protocols. Thank you. Galina Deyneko Novartis, Cambridge, MA 617-782-1675 home 617-871-7613 w --- ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Re: Parrafin tissues crumbles
Hi Michael. 1. Fixation : 48 hours at room T is a good time to fix a big rat heart. You can transfer at 4C after 24/48 hours, otherwise cold T from the beginning prevents good penetration and fixation. 2.. process the hearts and muscles separate from other tissues 3. use only fresh reagents in you processing machine. I always rotate reagents before process the hearts and this really works for me.2 hours in each Ethanols is OK in my opinion but according me experience it depend on processing center .I think that you use xylene substitute and I can recommend to switch back to real Xylene's. the clearing in Xylene is very short -30 Minutes?, especially after long dehydration. It would be better if you use 1.30 or 2 hours on each. Time in paraffin is OK, but I prefer 3 baths of paraffin, 1.30 or 2 hours in each. In the processing center use paraffin called infiltration medium i use from surgipath # 01400, for embedding i highly recommend Shandon Precision Cut paraffin from Thermo Shandon# B1002490. Do not freeze paraffin blocks this make the paraffin and tissues very fragile. You can cool the blocks in refrigerator or on the top of cold plate. Trim the paraffin block and place in the DI water for soaking for 5-6 minutes, do not keep to long. Use only DI water in the floating bath. Moisten the cutting surface of the block with your finger during sectioning, this is help to get more smooth sections. My English is not perfect but I hope you understand my tips.please contact with me if you have additional questions. Galina Deyneko Novartis, Cambridge, MA 617-871-7613 galinadeyn...@yahoo.com Message: 8 Date: Tue, 9 Nov 2010 14:56:49 -0800 From: Michael Mashore mmash...@vapop.ucsd.edu Subject: [Histonet] Paraffin Tissue Crumbles To: histonet@lists.utsouthwestern.edu Message-ID: 652586e049884d8491a0e70448595...@vandeberg Content-Type: text/plain; charset=iso-8859-1 Hello Histonet Users, I have just started using paraffin and am having many difficulties. Most of the time my tissue crumbles when sectioning. I have no real experience in paraffin histology and have been given the task of becoming proficient by myself, so I am hoping for feedback as to why my tissue keeps crumbling. The tissue in question has been: skeletal muscle, cardiac muscle, liver, and brain (all from rat). The tissue was fixed in 10% neutral buffered formalin for 7 days at 4°C and then transferred to an automated tissue processor, with the following schedule: 2 hours 70% dehydration alcohol 2 hours 80% dehydration alcohol 2 hours 95% dehydration alcohol 2.5 hours 95% dehydration alcohol 2 hours 100% dehydration alcohol 2 hours 100% dehydration alcohol 2 hours 100% dehydration alcohol .5 hour Hemo-De .5 hour Hemo-De .5 hour Hemo-De 1 hour paraffin 4 hours paraffin They were infiltrated for 1 hour without vacuum then embedded. The blocks were stored in the freezer before cutting. The knife angle was 5°. Sections were 5µm thick. I would appreciate any feedback whatsoever. Thank you very much. Michael -- ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] COX staining in frosen muscles
Dear Colleagues. Please help. I have been asking to perform staining for COX (which is cytochrome oxidase I believe) activity on mouse fresh frozen leg muscles. I am not very familiar with enzyme histochemistry. I found one method (Seligman,1968) in J.Bancroft , 5 edition book, and the other in J. Kiernan Histochemical Methods book, but the methods are described for the fixed brain. I am absolutely not sure that I have found the required methods. Could you share with me your experience, detailed protocol or the sources where i can find the reliable protocol siutable for muscle histochemistry.Thank you in advance. Galina Deyneko Novartis Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] succinate dehydrogenase protocol
Dear Colleagues I should detect /stain Succinate dehydrogenase in mouse muscle non-fixed frozen sections. Could someone please share detailed protocol and sources of the reagents. Does it exist any kit for detection? I performed some on-line search but did not find a sufficient information. Thank you in advance. Galina Deyneko Novartis, Cambridge, MA 617-871-7613. ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Insulin/Glucagon double IHC
Dear Colleagues, I would like to ask your advices. I should establish double IHC staining on FFPE isolated mouse pancreatic islets for alpha and beta cells. I have very restricted amount of slides and I do not have possibility to test different antibodies and protocols. Could you please give me references what are the best antibodies for insulin and glucagon for mouse and for human (for the future study) Langerhans islets staining.I think for double staining it is better to use the antibodies from different species. If you could share with me the protocols, I mean some tips about antigen retrieval, antibody dilution and incubation time, I will really appreciate it. As a secondary antibody i use the polymers from Biocare. Thank you in advance. Galina Deyneko. Novartis, Cambridge, MA 617-871-7613 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
