I am using rabbit polyclonal antibody ab31163 to detect Nrf2 in striatum of rat brain. They are 10um cryosections. I am following this protocol:
-fixation with 4% PFA for 10 min
-wash 3 times with PBS 3 min each
-put PBS tween 0.1% BSA 3% triton 0.5% for 30 min
-put PBS BSA 2% for 30 min
-incubate the primary antibody in PBS tween 0.1% BSA 3% overnight at 4 degrees
- wash 4 times with PBS
-incubate with secondary antibody for 2 hours (A568 goat anti-rabbit)
-wash 4 times with PBS
-Mount the slides with DAPI
Hi Hisham Altoufaily
The abcam antibody Anti-Nrf2 antibody (ab31163) has reactivity only to human samples and does not have reactivity towards Rat and is just predicted to work. So it may or may not work.
* The recommended section thickness for cryosections is (30-40um). You can also try with 10um.
* If the animal brains were fixed in 4% PFA overnight and then gradient sucrose before sectioning. I would advise you to use Ice cold methanol for 10mins prior to staining instead of PFA.
* Washing should be done with 0.1% T-X100 in PBS.
* 1 % T-X100 for 10mins is sufficient to permeabilize the tissues.
* Blocking using 3% BSA or 5% NGS for 1hr.
Rest all looks good.
Hope it helps,
Thanks,
- Badges
- Science topic
- Similar topics
- Psychology