Histoclear

For H & E staining, sections of skin were fixed in 4% paraformaldehyde in PBS for 16 h before embedding into paraffin. Thin sections (7 µm ) were cut and processed in an alcohol series (50–100%) for 5 min, stained with H & E for 3 min, and then rinsed in tap water for 3 min, before mounting on glass slides with an embedding media of Histo-Clear (Merck & Co, New Jersey, USA). The sections were examined under the microscope (Carl Zeiss Inc., Microimaging, LLC, Thornwood, New York, USA). Masson’s Trichrome staining was performed for identification of collagen [36] . For immunohistochemical staining, excess tissues were trimmed down and 7 µm thick paraffin sections were used to detect different antigens. Type I collagen deposits were immunodetected according to the described protocol. Tissue sections were incubated overnight with the primary antibodies (1∶50 or 1∶100 dilutions for collagen, and 1∶200 for CD-68) at 4°C, washed three times in PBS, and then incubated with peroxidase-conjugated secondary antibodies (1∶1000 in blocking solution) for 5 min. Sections were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, California, USA) and stored at 4°C. The sections were examined by a Laser Confocal microscope (Olympus Imaging America Inc., Center Valley, Pennsylvania, USA) connected to software (Flow view, FV10.AS), version (2.0 viewer).

Adult pituitaries were fixed for 16–24 h at room temperature in 10% NBF (Sigma). The samples were dehydrated and stored in 70% ethanol at 4 °C until they embedded. Wax embedding and sectioning were carried out as previously described [49 (link)]. Histological sections of 8 μm were used throughout. Slides were deparaffinized in Histoclear (Merck) and rehydrated through a descending graded ethanol series. Antigen retrieval was performed in citrate retrieval buffer pH 6.0 using a Decloaking Chamber NXGEN (Menarini Diagnostics) at 110 °C for 3 min. For paraffin immunofluorescence, sections were blocked in blocking buffer (0.15% glycine, 2 mg/ml of BSA, and 0.1% Triton-X in PBS) with 10% sheep serum (donkey serum for goat SOX2 antibody) for 1 h at room temperature, followed by incubation with primary antibody diluted in blocking buffer with 1% serum at 4 °C overnight. Slides were washed in PBST (0.1% Tween 20 in PBS) and incubated with appropriate biotinylated secondary antibodies or non-biotinylated secondary antibodies in blocking buffer for 1 h at room temperature. Slides were washed in PBST. Slides with biotinylated secondary antibodies were incubated with fluorophore-conjugated Streptavidin (Life Technologies) for 1 h at room temperature together with Hoechst (Life Technologies). After washing in PBST, slides were mounted with VectaMount (Vector Laboratories).

The tissues processed on a tissue array slide (BC081120f, US Biomax Inc, US Biomax Inc, Rockville, MD, USA) were deparaffinized and rehydrated in HistoClear and serially diluted ethanol (Merck, Darmstadt, Germany). After heat‐induced antigen retrieval, tissues were blocked with normal horse serum for 1 h and incubated overnight at 4 °C with primary antibodies. OCT4 (1 : 400; Abcam) and p62 (1 : 200; Santa Cruz Biotechnology) antibodies were used. Next, nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma‐Aldrich) for 15 min and 2 h at room temperature with Alexa Flour® 488‐ and 594‐conjugated secondary antibodies (1 : 1000; Enzo Life Sciences). The slide was mounted with a cover slide using mounting solution (Dako; Agilent Technologies, Santa Clara, CA, USA). Images were obtained with confocal laser scanning microscopy (LSM 700, Zeiss, Oberkochen, Germany) using identical exposure settings. The number of puncta was measured using i‐Solution software (version 22.5, InnerView™, Seongnam, South Korea). The intensity of nuclear OCT4 fluorescence was measured using imagej [18 (link)].