PREPERATION OF RENIN SUBSTRATE

ALL GLASSWARE MUST BE SILICONIZED OF PLASTIC

SOLUTION REQUIRED:

        1) SODIUM AZIDE (0.2g%)........ 20 mg/10ml OF DH2O
           -THE SOLUTION IS STABLE FOR A WEEK. LABEL AND DATE IF KEPT IN 
                REFRIGERATOR.
        2) PHOSPHATE BUFFER pH 7.0 FOR 250 ml EACH OF SOL.A AND SOL.B

                -SOLUTION A: Na2HPO4 60mM/..........2.1294g
                 EDTA 2.4 mg/ml.....................0.6000g  250ml
                 SODIUM AZIDE 20mg/L................0.0050g 

                -SOLUTION B: NaH2PO4.H20 60mM/L.....2.0748g
                 EDTA 2.4 mg/ml.....................0.6000g  250ml
                 SODIUM AZIDE 20mg/L................0.0050g

        SHAKE SOLUTIONS A AND B UNTIL ALL CHEMICALS ARE IN SOLUTION.
        (REMEMBER EDTA TAKES TIME TO DISSOLVE IN SOLUTION)

        PLACE A BEAKER WITH MAGNETIC STIRRER, UNDER pH METER.

        ADD SOLUTION B TO SOLUTION A UNTIL pH 7.0 IS REACHED IN A VOLUME
        WHICH IS SUFFICIENT TO DILUTE ALL THE PLASMA. (SEE BELOW)

        DISCARD A AND B THAT REMAINS; KEEP PHOSPHATE BUFFER pH 7.0 IN 
        REFRIGERATOR.

OBTAINING THE SERUM:

        A) NEPHRECTOMIZE 30 TO 50 RATS UNDER PREFERRED ANESTHETIC .
        B) 16 TO 24 HOURS LATER, ANESTHETIZE  AGAIN AND BLEED COMPLETELY INTO 
           PLASTIC CENTRIFUGE  BOTTLES, SET ON ICE. TO BLEED, EITHER ASPIRATE
           THE BLOOD FROM THE AORTA  WITH A PLASTIC SYRINGE AND NEEDLE OR 
           CATHETERIZE THE AORTA AND BLEED DIRECTLY INTO PLASTIC BOTTLE.

                KEEP BLOOD AND PLASMA IN ICE (4ø C) AT ALL TIME.

        C) CENTRIFUGE THE BLOOD FOR 20 MINUTES AT 800 G (2,600 SS34 SORVALL)
           IN REFRIGERATED CENTRIFUGE.
        D) SEPARATE THE SERUM FROM THE RED BLOOD CELLS AND MEASURE THE VOLUME.
        E) TO 9 PARTS OF SERUM ADD 1 PART OF SODIUM AZIDE (SOLUTION 1)
        F) DILUTE THE PLASMA-SODIUM AZIDE MIXTURE 1:5 IN PHOSPHATE BUFFER
           pH 7.0 (1 ml OF PLASMA + 4 ml BUFFER) USE REFRIGERATED BUFFER.
        G) MIX WILL AND SEPARATE IN POLYSTYRENE TUBES: 2ml IN ABOUT 1/2 THE 
           TUBES. 5 ml IN OTHER TUBES.

           LABEL THESE TUBES :  RENIN SUBSTRATE OR RS AND INDICATE VOLUME AND
           FREEZE.