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.

Add dH2O to bring up to 600 mL to make 0.1M PB. Check pH to make sure it is 7.4.