Periodate-Lysine-Paraformaldehyde (PLP) fixative for immunofluorescence staining (8-10-2011 Yujiro Kida)
(1) Solution A (Make ahead and freeze at -20oC)
Dissolve 7.3g L-Lysine monohydrochloride into 200 mL of dH2O.
Add enough 1M Na2HPO4 (MW: 141.96) to bring pH to 7.4.
Add enough 0.1M phosphate buffer (PB, pH 7.4) to bring the final volume to 400 mL* (*See below. PB is different from phosphate buffered saline (PBS).).
Aliquot into 18.75 mL and freeze at -20oC until day of use.
(2) Solution B (Make on day of use)
To 5 mL PBS, add 1 drop (50 µL) of 10N NaOH, and 0.4 g paraformaldehyde (PFA) to 15 mL tube.
Heat at 95C for 3-5 minutes then vortex to dissolve PFA.
Prepare PLP fixative
Thaw one aliquot of Solution A and add Solution B.
Add 51 mg of NaIO4 (sodium periodate, Fisher Chemical, S398-50, MW: 213.98) and dissolve it.
Keep it on ice. It is good for one use only (Use approximately 4 mL/ half kidney).
Immerse tissue samples in this fixative for 2 hours at 4oC.
Transfer samples to 18% sucrose dissolved in PBS and leave them overnight at 4oC.
Embed samples into O.C.T. compound after wiping out 18% sucrose/PBS.
*0.1M phosphate buffer (PB)
Make a stock of 0.2M NaH2PO4 (monobasic, MW: 119.99).
Make a stock of 0.2M Na2HPO4 (dibasic, MW: 141.96).
Mix 57 mL of 0.2M NaH2PO4 and 243 mL of 0.2M Na2HPO4.