Till date we used methanol acetic acid based fixative on cryosetioned (5 micron sections) human tissues and stored them at -20, so that it can be used for Florescent In situ Hybridization, Now we are planning to do IHC on same sections, but methanol leads to protein denaturation so we can't use it.
We are looking for a fixative which will be best for FISH and IHC on frozen tissues.
Dear Priyanka Bhosale i hope the following link will be helpful for your research
http://www.springerlink.com/content/w054v245ug471657/#section=615375&page=3&locus=72
Most cases for protein studies researchers use 4% Paraformaldehyde, 4% Paraformaldehyde-1% Glutaraldehyde or 10% Neutral-Buffered Formalin (NBF) and Picrates fixatives are useful for biogenic and polypeptide hormones targets and Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE) are good in preserving protein antigens and also useful for enzyme detection
Using Medium-OCT will be more useful for antigens sensitive to chemical fixatives
Priyanka, I have used the following protocol successfully for detection of DNA, RNA, and proteins from the same tissue.
1. fix the tissue in 10 volumes of 4% Paraformaldehyde for up to 4 hours @ room temp (shorter times for smaller samples). - do not exceed 4 hrs.
2. transfer the samples to 30% sucrose in phosphate buffer and leave at 4C overnight (tissues will float initially, but leave them in till they sink)
3. remove the tissues and embed in OCT. Store blocks at -80C in a plastic pouch to avoid dehydration over time.
4. Sections can be stored at either -20C long term before processing.
5. Antigen retrieval protocols are similar to frozen sections (optimized for your protocol/Ab).
Good luck!
Pyriyanka, if methanol is too strongly denaturing for your use, it seems unlikely that paraformaldehyde / formalin will work. In our experience this is stronger denaturing that methanol. we worked sucessfully with acetone fixation for about 2 minutes at room temperatue or - best results - 5 minuteswith -20C acetone. Air dry or gently use a hair dryer to remove acetone before proceeding with whatever water based buffer you continue.
Dr. Harald, I had a query, is acetone based fixation compatible with FISH? as I am planning to do FISH and IHC on same tissue
Dear Pyriyanka, acetone fixation is VERY mild - especially at -20c (minus 20 grade Celsius) - mostly you remove fat. I assume you will work with oral cancer and normal tissue, there it should work. I would expect problems in "agressively autolytic" tissues, e.g. exocrine pancreas by endogenous proteases.
I assume you will use fluorescence for IHC? With peroxidase you may need a methanol step anyway!
If FISH and IHC in the SAME section proves technically challenging, you may consider serial sections.
Priyanka,
If you are already finding problems with methanol based fixation and antigen detection, acetone will certainly not be much help.
To correct Harald - Paraformaldehyde (PFA)/formalin (aldehydes) are actually crosslinking agents and not denaturing agents. Numerous studies have shown that aldehyde crosslinking maintains activity of simple enzymes (reporters) and fluorescent proteins unlike denaturing agents like alcohols and acetone which deactivated these proteins by denaturation - structural. A simple search will yield a few hundred references for this.
That said, formalin and PFA are not without their faults - excess crosslinking will mask your epitopes, so you would want to scale back on the exposure time of the tissues to these agents - a mild fixation followed by OCT embedding will give you all the advantages of frozen and FFPE sections without exposure to denaturing alcohols/organic solvents.
thank you amit for your sugession,
Thats what the problem i was facing when i used FFPE tissues, Formalin fixed protein very well but for FISH was a problem hybridization didnt work; And wen i use methanol i get beautiful results for FISH but them nothing for IHC. So i planed of fixing cryosectioned tissues with either PFA/formalin(without parafin embedding) and acetone and look for both FISH and IHC. I hope either of this methods work for both my techniques. As i have got very small oral cancer patient samples, so need to use them very cautiously.
- Badges
- Science topic
- Similar topics
- Biological Science
- Histology