I am getting difficulty in getting cell marked distinctly.
I am using mouse model with the following protocol:
Pre exposure habituation in the chamber - 20 minutes
Exposure - 15 minutes
Post exposure silence - 60-80 minutes
I will be grateful if anyone can provide a better direction. Thanks in advance.
I have attached a very efficient protocol that I use to stain c-Fos positive neurons following a 90 minute post exposure silence period. The brain sections are 50um and fixed in 4% PFA via transcardial perfusion.
Thank you so much for your time and hearty help. I will surely follow the mentioned protocol. I have already undertaken the measure of PFA perfusion and using 50um sections.
Hope this will work good. WIll update about the outcomes.
Recently have got success using this short and customized protocol after doing few sets of trials and errors.
In brief: 1. after sectioning the tissue is kept in 4% PFA for 24-36 hours. 2. Wash 3 times with 1x PBS 3. Block in 2% NGS in 1X PBST in room temperature for 1 hr 4. Primary antibody (synaptic system, dilution 1:2000) in 1x PBST (0.3% Trition x 100) with 0.15-0.2% of NGS or in antibody solution. 5. Keep the sections floating in primary (1.5 ml primary in each well) for 72 hours in 4 degree, with constant slow shaking in rocker for better expression and for preventing antibody clumping. 6. Wash the sections in 1x PBS three times 7. Incubate in secondary antibody in 1x PBST with 0.2% NGS or antibody solution for 1 hour. 8. Wash the sections with 1x PBS for three times (15 minutes each) 9. Mount the sections on slides with flouromount with DAPI. 10. Kept overnight for drying. 11. Done - got expression marked.
The next batch of IHC staining are ready and yet to get the images. Hence, will update if any sort of update of the protocol is performed. Thank you.
Amiyangshu
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models
- Mouse Models