Hello all,
I am trying to detect cell death in the brain of adult zebrafish on sections. I performed immunohistochemistry on brain sections using the Purified Rabbit Anti- Active Caspase-3 antibody (BD Pharmingen 559565 ) with antigen retrieval, however, I did not see any positive staining. Any advice about this matter would really help me!
Thank you!
Dear Sam!
Please You look at this article and protocol:
Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons
https://dev.biologists.org/content/develop/143/22/4279.full.pdf
Expression analysis For whole-mount in situ hybridization, larvae were fixed in 4%PFA/PBS overnight and processed according to an established protocol (Volkmann et al., 2010). To detect Venus cleaved Caspase 3 by immunohistochemistry, embryos at 1 dpf were fixed in 4%PFA/PBS 6 h after treatment with EtOH or 4OHT. Larvae at 4 or 5 dpf were fixed after 4OHT treatment in 4%PFA/PBS for 3 h or overnight. For sectioning, brains from adults older than 15 months were fixed with 4%PFA/PBS overnight at 4°C followed by cryoprotection in 20% sucrose/PBS. After embedding in Tissue-Tek (OCT), cryotome sections were sectioned at 20 µm. The following primary antibodies were used: polyclonal rabbit IgG cleaved Caspase 3 (1:2000, Abcam, ab13847), polyclonal chicken IgY GFP (1:1000, Aves labs, GFP-1020), monoclonal mouse ZebrinII (1:300, a kind gift from Richard Hawkes, ACHR, University of Calgary), polyclonal rabbit IgG tRFP (1:2000, Evrogen, AB233) and monoclonal rabbit Caspase 8 IgG (EPR17366) (1:2000, Abcam, ab181580). The following secondary antibodies were used: donkey anti-chick IgY FITC (1:1000, Jackson ImmunoResearch, 703-545-155), donkey anti-rabbit IgG Alexa647 (1:1000, Thermo Fisher Scientific, A-31573), donkey anti-rabbit IgG Alexa546 (1:500, Thermo Fisher Scientific, A-10040) and donkey antimouse IgG Alexa488 (1:500, Thermo Fisher Scientific, A-21202).
If you only need to evaluate cell death without specifically defining apoptosis, TUNEL staining is always much easier to perform in zebrafish as it is chemical and not an antibody-based detection. TUNEL binds to the DNA breakup sites in the dead cells. There are DAB and fluorescence-based kits for TUNEL staining.
I agree with Anton Lennikov , TUNEL staining might be the best option , to evaluate the cell death ,but their might be chances that ur IHC protocol is not working , u can try different dilutions of antibody and can increase incubation time .
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