Table 3 Reagents and solutions used for sample preparation.

From: Serial scanning electron microscopy of anti-PKHD1L1 immuno-gold labeled mouse hair cell stereocilia bundles

 

Reagent

Final solution

Buffers

Leibovitz’s L-15 medium, no phenol red (GIBCO, 21083-027)

Hank’s Balanced Salt Solution, with calcium, magnesium, no phenol red, 500 ml (GIBCO, 14025092)

Hank’s Balanced Salt Solution, no calcium, no magnesium, no phenol red, 500 ml (GIBCO, 14175095)

Sodium Cacodylate Buffer, 0.2 M, pH 7.4, 500 ml (EMS, 11652)

Sodium Cacodylate Buffer, 0.1 M, pH 7.4

Fixatives

16% formaldehyde, 10 ml (EMS 15700)

4% formaldehyde, in HBSS with calcium, magnesium, no phenol red (pH 7.2)

2.5% Glutaraldehyde in 0.1 M Sodium Cacodylate Buffer, pH 7.4, 10 ml (EMS, 15960)

2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 1% tannic acid and supplemented with 2 mM CaCl2

Tannic Acid, Reagent, A.C.S., 100 g (EMS, 21710)

Blocking solution

Goat serum, 10 g

(Jackson Immunoresearch, 005-000-121)

10% goat serum in HBSS, no calcium, no magnesium, no phenol red (pH 7.2)

Antibodies

Rabbit anti-PKHD1L1 antibody, (NovusBio, NBP2-13765)

1:200 dilution in 10% goat serum blocking solution

Gold Conjugate EM Goat F(ab′)2 anti-rabbit IgG:10 nm gold

(BB International #14216)

1:30 dilution in 10% goat serum blocking solution

Staining solutions

Potassium Ferrocyanide Trihydrate, 50 g (EMS, 26604-01)

1% osmium tetroxide/1.5% potassium ferrocyanide in 0.1 M cacodylate buffer, (pH 7.2)

4% aqueous osmium tetroxide, 5 ml (EMS, 26604-01)

Dehydration and embedding solutions

Ethanol, 200 proof.

30%, 50%, 80%, 100% ethanol in distilled water

Propylene oxide, 450 ml

(EMS, 20401)

100% Propylene Oxide

Araldite-EMbed + propylene oxide (1:3)

Araldite-EMbed + propylene oxide (1:1)

Araldite-EMbed + propylene oxide (3:1)

Araldite-EMbed (Mollenhauer’s Kit): EMbed-812, 25 ml,

Araldite 502. 15 ml,

DDSA; 55 ml

DMP-30, 1.5–1.9 ml

(EMS, 13940)

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