Immunofluorescence method by using formaldehyde
Aim : To observe, in situ, yeast's proteins with tagged antibodies.
1 M potassium phosphate buffer at pH 6.5 (PPB6.5) :
Prepare 500 ml of 1 M potassium phosphate monobasic and 500 ml of 1 M
potassium phosphate dibasic. Mix both buffers until the pH 6.5 is reached.
Make 500 ml of that. Autoclave and store at Room T°C.
PPBMG pH 6.5 : 500 ml
1 M PPB at pH 6.5 60 ml for a final concentration of 120 mM
DdH2O 440 ml
Autoclave and store at Room T°C.
Formaldehyde buffer 5% (make fresh)
Add 13.5 ml of 37% formaldehyde to 86.5 ml of PPBMG pH 6.5.
Check the pH before using. If the pH is wrong, check the pH of the buffer. If
that's correct, check the formaldehyde. If it is off, just order a new bottle.
Nota bene : Formaldehyde 37% solution Microscopy grade (500 ml) from Sigma
(page 435 F1635) is at pH 5-6. Use until polymerization occurs.
Buffer B : 100 ml