[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

[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

[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.

[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

[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

[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

[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

[Histonet] Still having Issues with Acetone fixation.

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

[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

[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

[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

[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.

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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.

[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

[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

[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

[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

[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,

[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

[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

[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

[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?

[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

[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)

[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.

[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

[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.

[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?

[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

[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

[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

[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

[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.

[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

[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,

[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