It is better to fix your sample before IHC, because fixation can preserve component of cell and cell morphology. Fixation by alcohol usually use for preserve the genomic content and  detecting antigen in nuclei. Alcohol only precipitate antigens, it does not cross-link them. So some antigens may be washed out during IHC process, especially the cytosolic antigens. You can fix your samples by 4% PFA + 1% Acetic acid in PBS at RT for 10 to 30 min after preparing your slices (the thickness should less than 30  micron). After fixation, wash slices by cold PBS 10 min for three times at 4 degree. Under this condition, you can preserve cell morphology very well, and short-term fixation will not mask antigen too much.

It is better to fix your sample before IHC, because fixation can preserve component of cell and cell morphology. Fixation by alcohol usually use for preserve the genomic content and  detecting antigen in nuclei. Alcohol only precipitate antigens, it does not cross-link them. So some antigens may be washed out during IHC process, especially the cytosolic antigens. You can fix your samples by 4% PFA + 1% Acetic acid in PBS at RT for 10 to 30 min after preparing your slices (the thickness should less than 30  micron). After fixation, wash slices by cold PBS 10 min for three times at 4 degree. Under this condition, you can preserve cell morphology very well, and short-term fixation will not mask antigen too much.

Thank you chao. I am going to isolate DNA ( And RNA ,but not just now) after laser capture micro dissection. That is why I am trying to avoid use of PFA. however there are some literature regarding DNA isolation after use of PFA. Anyway I guess as you mentioned Alcohol can wash out some of the antigen, that could be the reason why my alcohol fixed sections are not giving any signals. Next I am going to try acetone and see if that works.  And if nothing works then I will go with PFA. Thank you once again.

Hi! Acetone has similar function like ethanol, so I am afraid you may get similar result like alcohol fixation. If you have enough samples, please separate your experiment by two protocols. For DNA and RNA purification, you can use 100% alcohol + 1% Acetic acid for fixation. For IHC, please use PFA for fixation. If you need Tau tangles to be a marker for LMD, I am afraid PFA will be the best way for you.

I agree with Chao, the advantage of human brain samples is that often you have enough issue to work with, having consecutive separate samples for molecular and for histopathology and IHC is good idea. You can also explore the option of antigen recovering methods, there are several in my experience microwave antigen recovering is the one that has worked the best for  PFA fixed tissues even after long term storage of the samples. Histopathology resolution is best on PDF fixed tissues. Here I enclose a protocol in case you want to try.  Regardless the fixation method  If you are not getting any signal is important to test antibody dilutions, do a dilution curve. and the thickness of the samples try changing the thickness.  

Microwave antigen recovery method Paraffin embedded sections (5-6 μ thickness) are mounted on charged slides (Surgipath, Winnipeg). Sections are deparaffinized and passed through a series of 100% ethanol. Endogenous peroxidase sites in the tissue are blocked in a 0.5% hydrogen peroxide/100% ethanol solution, then rinsed in 95% ethanol and double distilled water. The slides are transferred into coplin jars of 10 mM sodium citrate buffer (BDH), pH 6.0 and then microwaved for two 3 minute periods at 450W with gentle agitation. After cooling to room temperature, the slides are processed for immunohistochemistry as follows:

Hello Mitesh, 

If you are considering LCM, avoid fixation or used mild fixatives like ethanol or acetone. For immunostainings you should be fine with acetone as suggested by Chao. We tend to avoid PFA as it crosslinks proteins and many of the antigens become undetectable under regular immunodetection. 

Best

Hi,

If you are interested in TAU expression, you can do the IHC, even without fixation. Your samples are most likely full of ice crystals, caused by the snap freezing.  Thus fixation after cutting will not  correct them.  In other words, the morphology of your samples is already bad.

I will try IHC without any fixation. I don't think you need any cross-linking (formaldehyde) if you're only interested in TAU. Some small proteins can be washed away (S100 family), so there is need to cross-link (fix with additive fixatives, like formaldehyde but not coagulating, like ethanol) before frozen sample IHC.

Cheers,

Ivan

Try with no fixation following cryosectioning. Then, you can allow to dry the tissue on the slides (Superfrost +) or immediately place in the buffers for staining.  Sections may dislodge using the latter.  Stain, dehydrate and mount the sections.  The morphology will likely not be too great but you should see antigen.  Some antigens are VERY sensitive to various fixatives so you need to explore, but no fixation also works. Once had a huge project where it was necessary to cut sections and immediately stain without dehydration. We had tried acetone, acetone plus CH2O, Ch2O alone and the only thing that preserved antigen was unfixed hydrated sections as above. Bonne chance.

Dear Mitesh, This should be possible and you can combine with a cell membrane-permeant DNA counterstain - an example of this would be DRAQ5 or CyTRAK Orange.

The thin frozen sections can be laid down onto glass, dried down and stored at -80c until needed and then stained as described above. Best wishes. 

Dear ALL

Thank you so much for your valuable suggestions. looked like the system I was using didn't work for me( Primary+ Super Picture HRP polymer conjugate+DAB). However when I switched back to more orthodox way( Primary+ Biotin conjugated Secondary + ABC kit + DAB chromogen)  It worked. Moreover the fixing agent didn't matter that much as they all worked fine. If  have to give them a preference the order would be ACETONE, ALCOHOL, ACETONE+ METHANOL, and last Non foxed as all of them gave me good signal.

Again thank you all very much

Dear Patel,

You can use a mild fixative called Froz Fix.The fixative is designed for IHC on frozen sections. For IHC purposes you will need some fixative because some of the proteins leached into solutions when not fixed, I suggest to go to www.newcomersupply.com then go to fixatives, then to Frozfix. Click on camera icon. there is tutorial on how to use the fixatives as well prepare your frozen sections for IHC. The turtorial presents detailed steps from cryo, sectioning, conditioning of sections before IHC. I am aware of some publications using Froz fix bothe for routine chromogen IHC, Flourescence IHC as well as double labeling. I notice you are working with brain tissues, I suggest to cut thickness to around 6-10 microns thick.

Good Luck,

loui Harkins

follow up on frozen sections.

May be it will be worth trying smooth muscle action on bird brains ?

I was able to utilize frozen tissues in OCT chunks  about 6 years and older. Frozfix chemistry is designed to preserve morphology of frozen sections while maintaining level of antigenicity detectable to most proteins as well as dna and rna targets. I highly recommend the video tutorial presented at www, newcomersupply. com.

Good Luck and hope it well help further the knowledge in Immunohistochemistry.

Loui harkins

Dear all,

the response about actin is for another inquiry: on avian brains and not answer to Mr. Patel"s questions. sorry for wrong post.

Hello

# Yes you can use the frozen tissues in OCT . But , frozen tissues can be affected by the formation of ice crystals, which can affect subcellular detail and impact IHC staining. Another potential drawback to frozen tissue is the thickness of the section. Compared to paraffin-embedded sections .

# Usually after cryopreservation , slides are removed from the freezer and allowed to warm at room temperature. The slides are then immersed in fixative (usually alcohol or acetone) .you need choose the appropriate antibody for the frozen tissue , I recommend visit the R&D syestem , abcam and novusbio to more info .

Good Luck

Hello! Well, the best is submit samples to glycerine as cryoprotective and immersed the eppendorfs tubes in a mixture (1:1) hexane+liquid nitrogen.

Good Lucky

Hi there!

I'm used to work with flash-frozen human brain kept at -80ºC in the same way Patel do. As said before, freezing with liquid N2 doesn't help at all to preserve tissue structure, so depending on your goal for Tau staining that will be an issue.

I was using 10 um cryosections to perform laser microdissection after perfoming immunoFLUOrescence with NeuN antibody. In my case, I needed a quick staining to maintain proteins and use them in downstream applications, so some background was found (although neurons were clearly distinguishable, but maybe you can optimize the time and dilution of the antibodies).

I guess the protocol below can help you!

-    Fix 2 min in cold-acetone (directly, avoiding thawing of the slide!)

-    Dry 5 min at 37ºC

-    Delimit the area with PAP-PEN

-    Block 3 min with 1%BSA-0.25% Triton®X-100 in PBS-Tween (kept at 4ºC)

-    Incubate 10 min with the primary antibody at 37ºC (diluted in blocking solution)

-    Washing 3 times with PBS-Tween

-    Incubate 10 min with the secondary antibody, 37ºC at dark (in blocking solution)

-    Washing 3 times with PBS-Tween

-    Dry 10 min at 37ºC

Good luck!

Hi there Mitesh

Although it's not quite what you want to do you should have a look at the exciting new work from Tulane University that permits an H&E like staining without any processing of the sample, even from entirely fresh material to allow real-time histopathology.  This is on the cusp of allowing the pathologist to report to the surgeon on the quality of a biopsy of whether they need to go further to excise marginal tissue from a tumour.

See: Elfer, K.N., et al. "DRAQ5 and Eosin (‘D&E’) as an Analog to Hematoxylin and Eosin for Rapid Fluorescence Histology of Fresh Tissues." PloS one 11.10 (2016): e0165530.

Best wishes,

Roy

Back in the 80s when most IHC was performed on frozen sections I always used cold acetone to fix the tissue sections.  I also recommend embedding the tissue in OCT and solidify on dry ice, not liquid nitrogen.  I cut 8 micron sections.