BUFFERS AND SOLUTIONS USED IN THIS STUDY

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BUFFERS

Cacodyiate buffer (2.0 M)

Sodium cacodyiate, 3 H2<3 214 gm, distilled water 500 ml. The pH
was adjusted to 7.3 with HCI.

Carbonate bicarbonate buffer (pH 9.6)

Na2C03 1.6 gm, NaHCOg 2.9 gm, NaN^ 0.2 gm, distilled water 1000
ml.

Electrophoretic or electrode buffer (pH 8.6)

Tris 6.0 gm, glycine 28.8 gm, 10% SDS 10 ml. The volume was made
upto 1000 ml.

Phosphate buffer (pH 7.0)

M/15 Na^POjj 61.1 ml, M/15 KH-^Po^ 38.9 ml, distilled water 900 ml.

Phosphate buffered saline (pH 7.2)

Na^Po^ 2H20 13.52 gm, KH^o^ 3.26 gm, NaCI 11.6 gm. The volume
was made upto 1000 ml.

Phosphate-buffered saline (PBS) (pH 7.4)

Na^POjj 5.36 gm, KH^o^ 1.63 gm NaCI 5.8 gm. The value was made
upto 1000 ml.

Phosphate buffered saline glucose (pH 8.0)

Na2HPo({ (anhydrous) 13.48 gm, NaH^o^, 2 H20 0.78, NaCI 4.25,
glucose 1 gm, distilled water 1000 ml.

Phosphate-buffered glycerol
This was made by addition of 9 parts 87% glycerol and 1 part
phosphate buffer (pH 7.0).
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FBS - tween 20
This was made by addition of 0.05% tween - 20 with PBS (pH 7.4).

Sample buffer

1.5 M Tris HCI (pH 6.8) 0.5 mi, glycerol 0.5 ml, 10% SDS 0.5 ml,
Bromophenol blue (1%) 15 Ul, B mercaptoethanol 50 pi. The volume was
upto 5 ml with distilled water.

Tris-barbital buffer (pH 8.6)

Tris 4.45 gm, barbuturic acid 2.2 gm, calcium lactate 0.053 gm,
NaN3 0.065 gm. The volume was made upto 100 ml with distilled water.

Trans blot buffer (pH 8.6)

Tris 12.18 gm, glycine 57.67 gm, methanol 800 ml. The volume was
made upto 4000 ml with distilled water.

Tris saline buffer (TBS) (pH 7.4)

Tris 0.242 gm, NaCI 0.876 gm. The volume was made upto 100 ml
with distilled water.

TBS-tween 20

This was made by addition of 0.05% tween 20 with TBS (pH 7.4).

SOLUTIONS

Alcohol gel wash solution

Ethanol 46 ml, acetic acid 16 ml, glycerine 8 ml, distilled water 32

ml.

Destaining solution

Methanol 1000 ml, acetic acid 200 ml, distilled water 2800 ml.
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Embedding solution

Plastic mixture

Epon 81 2 25 ml

Araldite 6005 15 ml A

Dibutalphthalate 3 ml

Dodecenyl Succinic Anhydride (DDSA) B

Final mixture

Plastic A 11 ml

Plastic B 9 ml

1.5 to 2 drops/ml DMP-30 was added to plastic A and then plastic B

was added and mixed thoroughly.

Glutaraldehyde (3%) and paraformaldehyde (1%) solution

70% Glutaraldehyde 0.43 ml, 2.0 M cacodylate buffer 0.50 ml, 20%
paraformaldehyde solution 1.0 ml, distilled water 8.07 ml. The pH was
adjusted to 7.3 with IN HCI.

Glycerine and water solution

Glycerine 50 ml, distilled water 50 ml.

Lead citrate solution

Lead nitrate 1.33 gm, sodium citrate 1.76 gm. The ingradients were
placed in a 50 ml volumetric flask with 30 ml distilled water (glass). The
resultant suspension was shaken vigorously for 1 minute and was allowed to
stand with intermittent shaking in order to ensure complete conversion to
lead nitrate to lead citrate. After 30 minutes IN NaOH was added. The
suspension was diluted to 50 ml with distilled water and mixed by
inversion.

The staining solution was found to be of pH 12 + 0.1 (IN NaOH, 0.4 gm

of NaOH were dissolved in 10 ml of sterile distilled water).
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Paraformaldehyde solution (20%)

Paraformaldehyde 20 gm was dissolved in 100 ml distilled water by

heating upto 60-70°C and constant stirring. A few drops of 40% NaOH were
added and stirred again till the solution became clear.

Staining solution

Comassie Brilliant blue 0.4 gm, methanol 90 ml, water 90 ml, acetic
acid 20 ml.

Uranyl acetate solution (1%)

Uranyl acetate 1 gm, glass distilled water 100 ml.