Details of reagents required for Immunostaining.
|Reagent Type||Reagent constitution||Reagent Quantity (calculated)|
|Wash buffer||TBS + 0.025% Triton X-100||250 µL (0.25 mL) Triton X-100 in 1000 mL TBS|
|Permeabilization reagent||0.2% Triton X-100 in PBS||20 µL (0.02 mL) Triton X-100 in 10 mL TBS|
|Fixative||4% paraformaldehyde in PBS||10 mL of 16% PFA in 30 mL PBS|
|Antibody diluent buffer||TBS + 1% BSA||0.1 g BSA in 10 mL TBS|
|Blocking Buffer||10% normal serum + 1% BSA + TBS||1mL normal serum + 0.1 g BSA + 9 mL TBS|
|For double staining: 5% normal serum of species in which first secondary antibodies were raised + 5% normal serum of species in which second type of secondary antibodies was raised + 1% BSA + TBS|
|Note: Goat and donkey serum 60 mg/mL – Jackson Immuno Research|
|Buffers||1× TBS (10× TBS comprised of 500 mM tris base + 1.5 M NaCl + HCl to set pH to 7.4); 1× PBS||100 mL of 10× in 900 mL distilled deionized H2O|
BSA is bovine serum albumin, TBS is tris-buffered saline, PBS is phosphate-buffered saline.
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