11 November 2019 7 10K Report

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)

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