[Histonet] budget camera for DAB sections on an old olympus BH-2
Hi Everyone, I do a lot of DAB staining on relatively thick rat brain sections (40um). I have been using a really nice slide scanning system for publication quality images. Recently I acquired a really nice Olympus BH-2, it has great objectives pilfered from an old confocal microscope. I’m on the lookout for a used trinocular head so I can take pictures. We have two basic needs: 1) to provide a viewing screen so that more than one person can assess a section, going back and forth between 3-4 people becomes a pain. 2) I’d like to take mid-quality pictures for notebooks and to make decisions on which to image with more quality (so having my students send me pdfs of images). 3) it would be fantastic if I could use some sort of wi-fi system to use with phones/tablets, etc. So we could easily share photos without a computer. 4) cheap! My thought is to go with any one of the super cheap cameras out there that have nice sharing features or buy a used camera that’s decent. I’d like to keep it under $500. I’ve seen used Zeiss color Axiocams, olympus DP70s, 10, 11, 12s, and then various infinity, spot, qimaging and coolsnap cameras. Any suggestions for keeping things inexpensive, but taking decent quality images thats good for archiving, sharing and generally assessing the tissue to decide which section to image for publications? What I don’t know is what it would cost to get any of these used cameras working, do I have to pay for extra cards (firewire, etc), controllers, and software? Thanks! Caroline Bass Assistant Professor University at Buffalo ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] penetration problem with floating brain sections
Hello All, I’m doing some IHC with DAB on formaldehyde fixed rat brain floating sections, either 35 of 50 um thick. Usually I get fine staining of TH for example, but now I’m going after a neuron that's a bit sparse, and I noticed I have a couple of darkly stained cells. But I can see dozens of “ghost cells”, these appear to be very lightly stained in the right place. My guess is that the antibody isn’t penetrating fully, and so cells on the face of the sections are staining darkly, and some on the interior are just barely been stained. Any suggestions on improving penetrance? I usually have primary on overnight, shaking in the cold room, and secondary at RT for 2 hours. I was thinking of extending the primary to room temperature and maybe upping the triton-X concentration. Any other suggestions? Secondly, I noticed were getting some cracks in the tissue. We usually mount the DAB stained sections on some sort of tissue bond slide, and then let it dry overnight before dilipidating and coverslipping with permeant. My best guess is that sometime during the drying or coverslipping the tissue pulls away slight, resulting in these smallish cracks. Anyway, any advice on handling these two issues would be greatly appreciated. Thanks, Caroline Bass University at Buffalo ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] subbing slides for floating brain sections
Hi Everyone, I typically used charged slides to mount my 50 um floating brain sections. However, these seem pricey and I remember subbing slides in the past. I most do DAB staining, native fluorescence and occasionally immunofluorescence. Can someone tell me a good and easy protocol for cleaning and subbing slides? I currently have relatively old gelatin (7+ years?), Type B 225 bloom. Is this sufficient? A lot of protocols call for type A. Thanks! Caroline Bass University at Buffalo ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] problem with double IF of brain section
Hello everyone, I’m trying a double stain for GFP and tyrosine hydroxylase in floating brain sections. Rat, perfused with formalin. I’m having a big problem, in that it’s clear that the top side of the sections are well stained, as are the bottom, but everything in between is not. These sections are 50 microns. Any suggestions? I assume it’s a penetration problem. But I’m not very comfortable with IF in general. Here’s what my tech does: Placed sections each in a well of a 24 well dish and rinsed in PBS (to remove the Na azide), 1 X 5 min. Rinsed in PBS-0.5% Triton X-100, 3 X 10 min. Incubated tissue in PBST (0.25% Triton X) + 5% NGS, 1 X 60 to 120 min. Incubated sections in 1ary antibody in PBS-T(0.25%) overnight @40C (covered to minimized evaporation). Both primaries together. Tyrosine Hydroxylase 1: 4,000 ImmunoStar #22941 (mouse) a- GFP 1:2,000 Invitrogen 6455 (rabbit) Next day, rinsed tissue w/ PBS-T, 3 x 10 min. Incubated w/ 2ary ab : Alexa 555 donkey anti mouse 1:4,000, Alexa 488 goat anti rabbit, 1:2,000, 1 x 2hs @RT. 2ary ab diluted in PBS. Rinsed w/ PBS, 3 x10 min. Mounted on slides, coverslipped using ProLOng Gold w/ DAPI. Cured in dark place @ RT overnight. Thanks, Caroline ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Invitrogen A6455 rabbit anti-GFP dilution for immunofluorescence
Hi Everyone, I use Invitrogen/Life Technologies/Molecular Probes' antibody for GFP staining in PFA fixed rat brains. It works quite well in my hands at a concentration of 1:20,000 for DAB staining. However, I have need of immunofluorescence now and don't have a good starting point. The product sheet isn't very helpful and Invitrogen says to use the same Ab concentration which seems crazy. Almost everything I see shows a lower dilution with IF. Does anyone have experience with this particular antibody in the brain? If not, what's a good rule of thumb when trying to convert an antibody from DAB to IF? What would you suggest in terms of dilution? Thanks! Caroline ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] source for plus slides and labels
Hi Everyone, I'm trying to buy super frost plus slides (or color frost) for adhering brain sections. Fisher is very confusing on this, and I don't really remember buying slides for $1 each. Can someone recommend a source for plus slides that is cheaper? Also, I just started using labels for my rather low throughput immunos, I have a brady tls pclink printer, and the labels print well, but they didn't hold up to the xylenes. Can someone recommend a label for this printer? Any real world experience? Our protocol calls for overnight in xylenes. Finally, any recommendation on mounting media? I've always used per mount, but a lot of folks seem to be using xylenes free media. I'm doing mostly DAB immunos, so any advice is appreciate! thanks, Caroline Bass ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] protocol for EGFP IHC in frozen rat brains
Hello Everyone, I have some rat brains that should express GFP, the brains were collected fresh and snap frozen in dry ice/isopentane. They have been stored for several months in a -80. I'd like to collect 500 um sections, thaw mount them to a slide an reserve these for mRNA collection. I would then like to take intervening sections to stain for GFP. I have a staining protocol for perfused, formalin fixed, sucrose protected floating brain sections that works fine. I'm a little more concerned about moving to unfixed sections. I know I need to cut everything on a cryostat and I assume that I have to process the thinner sections on the slide instead of free floating. What is the best way to proceed? Here are some specific questions… 1) what thickness should I cut, 50 um works great for my fixed sections. 2) should I "fix" these sections by either paraformaldehyde vapors or dunking in fix? If so, what kind/concentration? 3) could I then proceed as though this were a normal GFP immuno, with DAB stain? 4) should I used plus slides or subbed slides? 5) will the sections stay on without fixation? Any and all suggestions would be appreciated! Thanks, Caroline ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] AO860 repair advice
Hi Guys, I'm taking apart my AO860 and have a couple issues. First is trying to pull apart the central shaft that the microtome lowers and raises on. I can get the primary parts away from each other, and pull off the gears associated with the handle, but the gear on the central shaft is a problem. Normally I would just ignore it but there is some gunk in there that is definitely catching. I'll try soaking in mineral spirits, but if that doesn't work I'd like to take the shaft off. The three units are conspiring against me. It looks like one pin, running perpendicularly through the shaft is holding it all together, but I don't know for sure. There is a top plate that prevents the bottom plate from coming off and then the gear prevents the bottom plate from coming off. I can move everything about 1 cm so hopefully the solvent can get in there. I'm just perplexed at this particular part. Should I be able to take it apart? Am I missing something? Here are some pictures… the middle one shows the top plate, bottom plate and pin in the shaft. The bottom picture shows the clearance I have in moving them all together. http://imgur.com/a/DkF3j The second thing is one screw is stripped and won't come off. This is a flathead small screw. It's not totally stripped, but it's stopped up enough that there's no way brute force can do it. Any advice? I've thought of supergluing the screw to the screwdriver, but I don't want to wreck my nice microtome! Thanks in advance, Caroline Bass University at Buffalo ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] an Olympus BH-2 trinocular
Hi Guys, Would you know where I could purchase a c-mount for an Olympus BH-2 trinocular head? I want to take some easy pictures of rat brain H&E sections with a 2X objective. I don't mind buying off of ebay, but I don't know what I need. Also, any recommendations for very inexpensive cameras? These don't need to be fantastic, it's more for documentation. Thanks, Caroline ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] used cryostat opinion
Hi Guys, I'm looking to section rat brain. A cryostat is not a huge necessity for my lab, I can borrow access from another. But it would be a nice item to have and I'm considering a few refurbished models, a Leica CM1800, CM1850 and CM1900. The difference in price between the 1800 and 1900 is $6500. Any suggestions here? Are there any features of the 1850 or 1900 that would really justify the leap in cost, considering that I will not be a heavy user? Thanks, Caroline ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
[Histonet] Microm HM 560 vacutome for brain sections?
Hello Everyone, I'm thinking about purchasing a cryostat. I will most likely section primarily rodent brains and I'm concerned about wrinkling. One rep suggested the vacutome system, which according to the literature "the patented vacutome stretches sections to eradicate artifacts". I was also considering the cryojane system, which I have seen in action and think it would work well. Though I may also obtain mRNA from these sections and I wonder how the UV curing part of the system could affect things. Does anyone have experience with the vacutome and can suggest whether this is a good way to go. Or is the Cryojane better? I'm also considering some used Leicas, from the 1800, 1850 and 1900. My needs are fairly minimal, it will get weekly and possibly daily use on occasion, but for the most part I don't need all of the bells and whistles, just something that can cut brain fairly well. Thanks, Caroline ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
