Hi,
May I know the recommended blocking buffers used in zebrafish immunofluorescence staining?
In one protocol it advices to use 3% BSA, 1% Triton X-100 and 1% DMSO in PBST (0.1% Tween 20 in PBS) but some mention to use 10% BSA in PBST.
And also how should I dilute the primary antibody? Usually we dilute primary antibody in the prepared blocking buffer. But some protocols mention to use 1% BSA in PBST even though they used 10% BSA for blocking.
Do I need to go through a blocking step before secondary antibody incubation as well?
Than you
Hello Dumindu Kavinda
1) I prepare blocking buffer for immunofluorescence that contain 5% normal serum + 0.3% Triton X-100 in 1X PBS. For instance, if I have to prepare 10ml of blocking buffer, I will add 0.5ml of normal serum from the same species as the secondary antibody + 30 µL Triton X-100 to 9.470 ml of 1X PBS.
Alternatively, you may use 1-3% BSA in place of 5% normal serum.
Also, you may use Tween-20 (0.05%-0.2%) in place of Triton X-100, the concentration of which may vary depending on the strength of the antibodies being used.
I would not recommend 10% BSA in PBST as blocking buffer.
2) Primary antibody dilution may be prepared in 1X PBS containing 1% BSA and 0.3%Triton X-100. I prepare my antibody dilution buffer by adding 0.1 g BSA and 30 µL Triton X-100 to 10 mL 1X PBS.
The use 1% BSA in PBST as antibody dilution buffer is right.
3) Do I need to go through a blocking step before secondary antibody incubation as well?
No, you will have to perform the blocking step only once just before adding the primary antibody.
Best.
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