I'm new to immunohistochemistry and there's one step I'm having problems with. Generally I can pipet volumes pretty accurately and avoid air bubbles, but:

1) The BSA solution that I made (with PBS and BSA crystals - then vortexing) had a lot of air bubbles that wouldn't settle over time

2) I couldn't avoid air bubbles in my pipet even though I placed the tip under the layer of bubbles

How should I improve my technique? (Also, will having slightly different concentrations of the blocking and primary antibody serums greatly affect the result?)

Thanks, L

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Never vortex the BSA solution. The best way to prepare a BSA solution is to put the BSA powder on the buffer solution (or water) instead of the other way round. Tap or rock the tube to dissolve the BSA. If there are some tiny clumps then pipette very gently (use 1ml tips; cut the tips if needed) but tapping/rocking should usually do the job.

From CSHL protocols:

For a 10% (100 mg/mL) stock solution of BSA, dissolve 1 g powdered Fraction V or molecular biology grade BSA in 10 mL of distilled H2O; to avoid clumping, dissolve by layering the powder on the surface of the liquid. Gently rock the capped tube until the BSA has dissolved completely. Do not stir. Store in aliquots at −20°C.

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