Hematoxylin solution

Dissected cochlear tissues were fixed by 4% PFA in 0.1 M PB (pH 7.4). After permeabilization with PBS containing 0.3% Triton X‐100 (PBS‐0.3T), fixed tissues were incubated with primary Ab for 2 h at 23°C in PBS‐0.03T and 0.5% fat‐free bovine serum albumin (BSA), followed by Alexa488‐conjugated secondary Ab for 2 h at 23°C, and mounted in Prolong anti‐fade (Invitrogen) with a coverslip. Immunostainings were observed under a LSM700 confocal microscope (Carl Zeiss). In the case of samples embedded in paraffin, after deparaffinization, HE staining was performed using hematoxylin solution and eosin solution (Muto Pure Chemicals). Samples were imaged under a light microscope (BX50; Olympus) with a DP26 camera (Olympus).

Forelimb buds and a square area of abdominal skin, including feather follicles, from staged chicken embryos were used for histology. Samples for HE staining were fixed with Bouin’s fixative (9% formaldehyde, 5% acetic acid and 75% saturated picric acid) at room temperature (RT) overnight with shaking. The samples were washed with 70% ethanol (with saturated lithium carbonate) for 5–7 days followed by dehydrating with 80%, 90% and 100% ethanol (twice for 100%). Then the samples were permeated with xylene at RT once, 1:1 xylene/paraffin (paraplast, Leica) at 45 °C once, and paraffin at 60 °C three times and embedded in paraffin at RT. The paraffin blocks were sectioned at 10 μm in thickness on slide glasses (MAS-coated, MATSUNAMI). The sectioned samples were immersed in xylene for 20 min and then quickly immersed in 100% (twice), 90% and 70% ethanol and then rinsed in water. The sections were stained with hematoxylin solution (Muto Pure Chemicals) at RT for 1 min and washed with water followed by eosin staining for 30 s and water washing. The samples were dehydrated with a series of ethanol (70%, 90% and 100% ethanol) and xylene and were mounted with Eukitt (ASONE).

Lung tissue sections were mounted on gelatin-coated slides. After immersing the sections in hematoxylin solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan; No. 3008-1) and eosin solution (Muto Pure Chemicals Co., Ltd.; No. 3200-2), sections were dehydrated in ethanol, cleared in xylene (247642; Sigma-Aldrich, Darmstadt, Germany), and coverslipped with Permount (SP15-500; Thermo Fisher Scientific, Waltham, MA, USA). The left lung volume, percentage of cell infiltration area, and air space were quantified by the H&E-stained left lung sections [17 (link),18 (link)].

Luxol fast blue (LFB) staining was performed according to the manufacturer’s protocol (LFB Solution; Muto Pure Chemicals, Tokyo, Japan). H&E staining was performed using hematoxylin solution (Muto Pure Chemicals) and 1% eosin in water (Wako Pure Chemicals). Immunofluorescence staining was performed using a Mouse-on-Mouse immunodetection kit (Vector Laboratories, Burlingame CA), anti-VAMP2 antibody (1:200, cl:69.1; Synaptic Systems, #104211, Goettingen, Germany), anti-cyclin D1 antibody (1:50, cl:SP4; Thermo Fisher Scientific KK, #RM-9104-SO, Yokohama, Japan), AlexaFluor488-conjugated donkey anti-rabbit IgG F(ab’)₂ fragment (1:500; Jackson ImmunoResearch, West Grove, PA), and Rhodamine RedX-conjugated donkey anti-mouse IgG F(ab’)₂ fragments (1:500, Jackson ImmunoResearch). Microscopic images were obtained using a Nikon AZ-100 (Tokyo, Japan) and a cooled CCD camera (SPOT RT3; Diagnostic Instruments, Sterling Heights, MI).

BM-MSC spheroids were fixed in freshly prepared PBS (−) containing 4% paraformaldehyde (pH 7.4) for 1 h and embedded in paraffin, using standard histological procedures. The spheroid blocks were cut into 8-μm thick sections and mounted on glass slides. For H&E staining, slides were deparaffinized with xylene and re-hydrated using an alcohol gradient of absolute alcohol, 95% alcohol, and 70% alcohol. The slides were then washed in distilled water and stained in hematoxylin solution (Muto Pure Chemicals) for 5 min. The slides were washed in running tap water for 5 min and counterstained in eosin Y solution (Muto Pure Chemicals) for 1 min. Stained slides were dehydrated using 70% alcohol, 95% alcohol, and 100% alcohol, and cleared in xylene twice for 5 min. The slides were then mounted in malinol (Muto Pure Chemicals).

Testes were fixed in Bouin solution, and 4 μm plastic sections were prepared using Technobit 8100 (Heraeus Kulzer). Samples were stained with hematoxylin solution for 10 min (Muto pure chemicals, Tokyo, Japan), and stained with 1% eosin (Muto pure chemicals, Tokyo, Japan) for 30 s. The mounted samples were imaged using an Olympus DP74 camera (Evident, Tokyo, Japan).