Intra-epidermal Nerve Fibers Density and Nociception in EPO-Treated Type 1 Diabetic Rats with Peripheral Neuropathy - Tissue-Protective Cytokines: Methods and Protocols - page 194

4. 0.1 M Lysine stock solution for Paraformaldehyde-lysine-perio
date (PLP) mix. Dissolve 1.64 g of L-Lysine in 30 mL water,
in a 150 mL graduated cylinder. Adjust pH to 7.4 with 0.1 M
sodium phosphate dibasic dihydrate (DIBA). Add water up to
45 ml. Add 0.1 M PB up to 90 ml. Store at 4°C for up to 3
weeks.
5. 2% PLP fix for skin biopsies (see Note 4). To 30 mL of lysine
stock solution add 0.085 g of sodium periodate powder. Shake
until powder dissolves (5-10 min). Add 10 ml of 8%
Paraformaldehyde stock solution. Keep at 4°C.
6. Cryoprotective solution. Mix 20% glycerol, 20% PB 0.1 M,
and 60% water. Store at 4°C up to 1 month.
7. Glycerol-based antifreeze solution. Mix 300 mL glycerol,
300 mL ethylene glycol, 200 mL PBS. Make up to 1 L with
water. Store at 4°C up to 1 month.
8. 0.25% potassium permanganate solution. Dissolve 25 mg of
potassium permanganate in 10 mL water. A slightly purple col-
ored solution is obtained.
9. Oxalic acid (OX) solution: Add 0.45 mg of oxalic acid to 9 ml
water and stir for 2 min.
10. Tris Buffered Saline (TBS). Dissolve 12.11 g Tris base and
9.0 g sodium chloride in about 900 mL of water, stir and adjust
the pH to 7.4 with HCl. Make up to 1 L with water. Store at
4°C up to 1 month.
11. Block solution: 4 mL normal goat serum and 100 μ L Triton

X-100; make up to 100 mL with TBS.
12. Quenching solution: 30 mL of methanol and 1 mL of hydro-
gen peroxide; make up to 100 mL with PBS.
13. 0.09 M sodium citrate Buffer: 0.53 g of sodium citrate and
.0.38 g of citric acid in about 15 mL of water, stir and eventu-
ally adjust the pH to 4.2 with NaOH. Make up to 20 mL.
Store at 4°C up to 1 month.
14. 10 mM Tris-sucrose buffer solution (see Note 5): 0.303 g of
Tris base, 21.4 g sucrose, and 0.12 g of EGTA adjust to pH 7
with HCl and make up to 250 mL with water.
15. Buffer A: 15.86 g Tris base, 0.399 g EGT, 7.4 g NaCl, 0.976 g
KCl, 0.341 g sodium azide in 1 L water. pH to 7.4 at 37°C
and adjust to final volume. Store at 4°C up to 1 month.
16. Solution A1: Dissolve 27 mg of phosphoenolpyruvate and
18.44 mg nicotinamide adenine dinucleotide (NADH) in
50 mL.
17. Ouabain solution: Dissolve 54.66 mg ouabain in 25 mL with
solution A1.