4、床。 12.取载玻片,滴加10uL抗荧光衰减封片剂,将爬片有细胞面盖在封片剂上,指甲油封片子的对角线。 All steps forIF1) Remove culture medium and fix cells (a common fixative is 4% formaldehyde in PBS, for 15 minutes) 2) Wash well in PBS (3 x 5 minutes is typical) 3) Permeabilize the cells (a common permeabilizatio
5、n reagent is 0.2% Triton X-100 in PBS for 30 minutes) 4) Wash well in PBS 5) (optional: Block for non-specific dye binding using the Image-iT FX Image Enhancer Solution, I36933) 6) Block for non-specific antibody binding 30-60 minutes (a common blocking solution wo
6、uld be 3-6% bovine serum albumin / 5% normal goat serum / PBS, or commercial blocking reagents like our BlockAid, product B10710) 7) Incubate in primary antibody for 30-60 minutes, in blocking solution or overnight at 4 degrees (antibody concentrations vary, but usually b
7、etween 0.5-10ug/mL) 8) Wash well in PBS 9) Incubate in secondary antibody for 30-60 minutes, in 3-6% bovine serum albumin / PBS (a good starting antibody concentration is 5 ug/mL) 10) Wash well in PBS 11) Counterstain as needed (such as with DAPI, D1306) 12) Mount in
8、appropriate mounting medium (for fluorescent secondaries, a good antifade s