After SNL i harvested the spinal cord segments, Using Trizol reagent i isolated RNA then Protein extraction, measured protein concentration by BCA and had a relatively good concentration. Used the protein for WB. Primary antibody was Iba1, i couldnt get any band. but i had very clear bands when i used GFAP=astrocytes.
On ther hand, after perfusion fixation with 4% PFA, harvested the spinal segment, overnight in PFA, followed by 10,15,20% sucrose. cryopreserved the sections. cut 30um sections and tried to staing Microglia, using the same Iba1, but failed.
Has anyone encountered a similer difficulty in both WB and IHC-F?
What did you do to solve the problem?
Will changing the company help?
Thank you.
Hi, Costansia.
My protocol is:
a. perfusion fixation with 4% PFA
b. 4 hours in PFA in 4℃
c. 20%, 30% sucrose
d. cut into 20 um sections
e. store the secitons in 30% (W/V) surose, 30% (V/V) ethylene glycol in PBS
f. IF staining with anti-Iba1.
I always got good images.
You can follow the IF protocol from Creative Diagnostics at:
https://www.creative-diagnostics.com/immunofluorescence-protocol-free-floating-section.htm
Hope it will be useful!
Hi Costansia,
Are you sure your anti-iba1 antibody is a good one? Here are two anti-iba1 antibodies working very well in my hands: one from Dako and the other from Millipore.
For western blot, the size of the band should be around 17 kDa. And if you are using normal tissues (no microglial actvation), you may need to load more. Iba1 protein level is not very high in the normal CNS tissues. Anohter thing is that, if it is possible, avoid to use trizol for protein extraction.
For staining, if you have a good antibody, just follow the standard protocol. Iba1 staining is not difficult to perform if your antibody is good.
I have had similar problems with imunos using Iba1. I ended up opting to study different microglia subpopulations using specific markers.
Feedback for this question. i used Iba1 from Wako. 1:500 to 1:1000 and it worked well. thank you so much for your answers.
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