I'm studying the distribution of collagen I and II across the intevertebral disc of the Sprague Dawley tail using IHC. The fluorescent signals were supposed to vary from one another and appear gradient-like (transition of bone to cartilage markers). However, for all 3 stains, they appear to be emitted all throughout the tissue. This is my 2nd attempt (my first didn't give any useable images as the tissues detach from the slides during antigen retrieval) and since I'm relatively new to the technique, may I know whether it is an issue with my protocol, antibody handling, or tissue processing? Here is a snapshot of my procedures.
Tissue processing
Tissue was fixed in 10% NBF, after harvesting from rat carcass stored at 4deg. over the weekend. Was kept in fixative for 18 days at 4deg then 28 days at rtp (didn't realise till later that fixation should be done at rtp).
Decalcified using 30% formic acid for 2 weeks then stored in EtOH for 2 weeks until embedded.
IHC Protocol
1. Dewaxing using xylene, dehydration using gradient series of EtOH
2. Antigen retrieval using 10mM sodium citrate buffered to pH 6.0, slides placed in microwaved-boiled buffer for 20mins and a further 30mins for cooling (prevent tissues from falling off) before proceeding to next step
3. Wash with 1x TBST (20mM Tris, 150mM NaCl, 0.1% Tween 20) for 5mins
4. Block with 1% BSA made with PBS for 1hr @ r.t.p in humidified chamber.
5. Add primary Ab(s) and incubate in humidified chamber overnight @ 4deg.
6. Pipette up Abs and wash sections with wash buffer 3 times for 5mins each
7. Add secondary Ab(s) and incubate for 50mins in opaque humidified chamber.
8. Pipette up Abs and add DAPI to stain nucleus.
Antibody
Aliquots of 10uL stored at -20deg.
Stains
1. Ms mAb to ColI (stains red), 1:500 dilution (final 1:1000) - lowest conc. suggested
2. Rb pAb to ColII (stains green), 1:200 dilution (final 1:400) - lowest conc. suggested
3. DAPI to nucleus (stains blue)
I would say over fixation is the main problem. I have never stained the tissue of your interest, but have worked with muscles and neural tissue (including whole brain and whole muscles). The max I have fixed is 24 hours. When I have overfixed, stained, and imaged, I got back similar images you are showing. Try reducing the fixation time.
Perhaps fix 24hrs, wash thoroughly, embed in OCT (+freeze), cryosection onto positively charged slide and then proceed with the staining procedure. This is the most basic protocol. Again, I can't speak for the tail tissue processing, but you have a lot of steps. I have only done antigen retrieval when the tissue was paraffin embedded. Sometimes this can also cause autofluorescence. Be sure you are washing your secondary antibodies as well. Any nonspecific staining will remain without washes (you may be washing but it is not included in your protocol above).
Best of luck!
Elliot
Elliot Glotfelty Thank you so much Elliot! Yes, I've been told that issues may arise from over fixation once I move from histology staining to IHC. Good thing I've since harvested and fixed the tissues for a shorter period. I'll work on those and present their results in due time :)
You could cut the tissue into smaller blocks and fix at RT. This allows faster penetration into the tissue. Also coat your slides with a suitable adhesive like albumin fixative or polylysine. Also how are you accounting for non-specific fluorescent? Try making up the primary ABs in a buffer with 5% serum of of the animal from which the second antibody was developed. Also don't cut the sections too thick or you run the risk of losing them during the procedure.
Sheldon R Gordon Thanks for taking the time to read my question Sheldon!
Yes I do section the spine into 2 caudal vertebrae with the IVD in between (since I'm studying the enthesis). I do use the Superfrost charged slides.
My diluent is PBS with 1% BSA, though the secondary Abs were developed in goat, therefore I should try 5% goat serum from your suggestion? I did mention this to my colleague previously who said it is better to use BSA as goat serum contains more proteins and therefore is unsuitable? Nevertheless, I'll try your recipe.
Lastly, how thick is too thick? My samples are 5 microns, and I do admit even after careful handling, the sections detach at places and overlap onto other areas of the sections.
5 micron sections should be fine. If the secondary AB was made in goat then use 5% normal goat serum to block nonspecific binding when incubating in the primary AB.
Sheldon R Gordon Thanks so much for clarification! Will make the necessary adjustments soon :)
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