Hello
I am staining rat paraffin embedded 7um thick coronal tissue and using a protocol that was shared with me that was working very well for the post doc who has now left. this worked a year ago. The tissue is of CCI 3day post injury and my protocol consists of this down below:
The secondary is the supersensitive kit https://biogenex.com/product/super-sensitive-polymer-hrp/?v=79cba1185463
This kit works for other people...so its not the SS kit.
Problem: I have no background, and the DAB takes ages to develop (30-45mins) and once it has developed it only stains the soma of the microglia and not the processes or arms. My quantification depends on them
No one in my lab knows why. I wondered if it were my antigen retrieval
I have tested different brains and different antibodies and similar or no results. I cannot figure out what is going wrong.
please advise,
Thank you!
Protocol:
SS Day 1:
1. Melt wax for 10-15 minutes at 60°C or overnight at 37°C
2. Deparaffinize and hydrate samples 2x Xylene 5 min, 2x 100% Ethanol 2 min,
1x 90% Ethanol 2 min
1x 70% Ethanol 2 min
dH2O 2 min
3. Wash: 2x with 1xPBS, 5 min/wash
4. Permeabilization/Quenching: 1% H2O2 in PBS-Tx 0.3% for 30 minutes – cover otherwise h202 breaks down in warm and sunlight
5. Wash: 1x with 1xPBS, 5 min
6. Prep steamer
7. Antigen retrieval: 0.01 M Citrate Buffer (pH 6) antigen retrieval - Steam for 20 mins (first ten are in steamer heating up)
Cool down to RT on ice, transfer to dH2O – must be fresh!!
8. Wash: 1x with 1xPBS, 5 min/wash
9. Primary AB: in PBS-T (0.3% Triton-X) overnight a 4°C,
1 section slide = 100 µl; 3 section slides ~200 µL
SS Day 2:
1. Wash: 3x with 1xPBS 5 min/wash
2. Super-Enhancer 20 min
3. Wash: 2x with 1xPBS 5 min/wash
4. SS-Label 30 min – make DAB just before next step and leave on bench wrapped in foil
5. Wash: 3x with 1xPBS 5 min/wash
6. DAB-buffer + DAB (1 mL of DAB-buffer + 2 drops of DAB)
7. Stopping: MilliQ water for 2 minutes
8. Wash: Tap water for 2 minutes running (binds the dye and removes excess DAB).
9. Counterstain: Mayer’s Haematoxylin check at 1 min (>5 min for rat tissue), destain in running tap H2O for 5mins. Check quality of counterstain on microscope
10. Dehydration: Dehydrate in Histology lab in ascending series of
EtOH: 70%, 90% and 2x100%: each 2 min
Xylene: 3x 5 min
11. Coverslipping: Mount immediately in DPX
DAB should develop quickly so it is not getting in. I would switch the order of your blocking/permeabilization and antigen retrieval steps. You want to retrieve the antigens first. Then block any non-specific sites and permeabilize. Permeabilization also breaks through some of the protein cross-links caused by fixation, increasing binding to the correct epitope and reducing non-specific hydrophobic interactions. You need to retrieve/unmask the antigens first then break the cross-links. In addition, I might do the peroxidase block alone for 5-15 minutes and then the protein block separately for 30 minutes.
Hazel G May Have a look at the troubleshooting guide here: https://www.bosterbio.com/protocol-and-troubleshooting/ihc-troubleshooting. Also, get a known positive control tissue and rerun. If it is repeated, that shows the problem lies in the reagents. Then, you can further troubleshoot down from the guide here. With curiosity, I am confused about your dewaxing time (10-15mins) stated above, are you sure the protein is exposed? This is why you may need a readily confirmed positive FFPE block, barring the same processing protocol, perhaps from a vendor or sister lab.
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