[Deparaffinization]
(1) Xylene down I: 10 min
(2) Xylene down II: 10 min
(3) 100% ethyl alcohol (= ethanol) down I: 10 min
(4) 100% ethanol down II: 10 min
(5) 90% ethanol down: 10 min
(6) Iodine alcohol down: 10 min (to eliminate the sublimate)
(7) 0.25% sodium pyrosulfite: within 1 min (to eliminate the iodine)
(8) Phosphate-buffered saline (= PBS) I: 5 min
(9) PBS II: 5 min
(10) PBS III: 5 min

[Immunocytochemistry]
(1) 0.3% H₂O₂ in methanol: 30 min (preincubation to inhibit endogenous peroxidase activity)
(2) Distilled water (= D.W.): 30 sec
(3) PBS I: 5 min
(4) PBS II: 5 min
(5) 1% bovine serum albumin-PBS: 2 h (incubation)
(6) Monoclonal antibody to bullfrog luteinizing hormone beta-subunit (LH beta) at a concentration of 1/10,000 (BL4B11, Park et al., 1987): overnight (incubation)
(7) Goat anti-mouse gamma-globulin serum (1/50 concentration): 2 h (incubation)
(8) Mouse peroxidase anti-peroxidase immune complex (1/50 concentration): 1.5 h (incubation)
(9) PBS I: 5 min
(10) PBS II: 5 min
(11) PBS III: 5 min
(12) Graham-Karnovsky's medium: 15 min (visualization of the final reaction product)
(13) D.W.: 5 min
(14) 0.2% methyl green: 30 min (counter-stain)
(15) Washing with running tap water: 20 min
(16) D.W.: 30 sec

Park, M. K., S. Tanaka, H. Hayashi, Y. Hanaoka, K. Wakabayashi, and K. Kurosumi. 1987. Production and characterization of a monoclonal antibody against the beta-subunit of bullfrog lutropin. General and Comparative Endocrinology 68: 82-90.

[Dehydration and Immersion]
(1) 70% ethanol up: 10 min
(2) 90% ethanol up: 2 min
(3) 100% ethanol up I: 3 min
(4) 100% ethanol up II: 5 min
(5) Creosote-xylene up: 5 min
(6) Xylene up I: 5 min
(7) Xylene up II: 10 min
(8) Mount of sections in 6-7 drops of a mixed solution of Canada balsam and xylene with a 24 x 45-55 mm micro cover glass

[Components of each reagent]
(1) Bouin-Hollande sublimate (100 ml): Mix Bouin-Hollande's solution (90 ml) with saturated sublimate solution (10 ml)
(2) Bouin-Hollande's solution: Dissolve copper acetate (2.5 g), ground minutely in a mortar, in D.W. (100 ml), and further dissolve picric acid (4 g) in this solution. After filtrating it, add formalin (10 ml) and glacial acetic acid (1 ml) to this solution
(3) Iodine alcohol: 70% ethanol with 2-3 drops of Jode Tincturia (= iodine tincture)
(4) Jode Tincturia: Dissolve I₂ (6 g) and KI (4 g) in D.W. (100 ml)
(5) Phosphate-buffered saline (PBS: pH 7.5): Mix 0.01M sodium phosphate with 0.14M NaCl, containing 0.01% Merthiolate (= thimerosal) as antiseptic
(6) Graham-Karnovsky's medium: Dissolve 3, 3'-diaminobenzidine tetrahydrochloride (45 mg) in 0.05M Tris-HCl buffer, pH 7.6 (100 ml), and then add 30% H₂O₂ (0.1 ml) to this solution
(7) 0.05M Tris-HCl buffer, pH 7.6 (100 ml): Dissolve Tris (605 mg) in D.W. (95 ml), and then add 1N-HCl (4.2 ml) to this solution
(8) 1N-HCl (4.2 ml): Add 37.2% HCl (0.35 ml) to some amount of D.W., and then increase D.W. up to 4.2 ml
(9) Creosote-xylene (200 ml): Mix creosote (50 ml) with xylene (150 ml)