Epididymal adipose tissue was collected from WT and Plap-1 KO mice after 16 weeks of feeding HFD and then fixed in 4% Paraformaldehyde Phosphate Buffer Solution (Wako Pure Chemical Industries, Osaka, Japan) overnight. Samples were embedded in paraffin and sectioned at 3.0 μm with LEICA RM2245 (Leica Microsystems, Wetzlar, Germany). Sections were stained with Mayer’s Hematoxylin (MUTO PURE CHEMICALS, Tokyo, Japan) and 1% Eosin Y Solution (Wako Pure Chemical Industries, Osaka, Japan). Stained sections were observed and imaged with ECLIPSE Ci (Nikon, Tokyo, Japan). The size of adipocytes was measured with ImageJ software.
Mayer s hematoxylin
Manufactured by Muto Pure Chemicals
Sourced in Japan, United States
Mayer's hematoxylin is a laboratory reagent used for staining biological samples. It is a nuclear stain that selectively binds to the DNA and RNA in cell nuclei, allowing for the visualization of cellular structures under a microscope.
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Lab products found in correlation
Eosin alcohol solution, by Muto Pure Chemicals (6 mentions)
Malinol, by Muto Pure Chemicals (5 mentions)
Avidin biotin blocking kit, by Vector Laboratories (3 mentions)
Hrp conjugated sheep anti dig f ab fragment antibody, by Roche (2 mentions)
Hrp conjugated streptavidin, by Agilent Technologies (2 mentions)
Horseradish peroxidase, by Roche (2 mentions)
Formalin neutral buffer solution, by Fujifilm (2 mentions)
Chloral hydrate, by Nacalai Tesque (2 mentions)
Diff quik, by Sysmex (2 mentions)
Oct compound, by Sakura Finetek (2 mentions)
Envision hrp kit, by Agilent Technologies (2 mentions)
Peroxidase conjugated streptavidin, by Nichirei Biosciences (2 mentions)
Envision flex, by Agilent Technologies (2 mentions)
Eosin y solution, by Muto Pure Chemicals (2 mentions)
Paraformaldehyde phosphate buffer solution, by Fujifilm (1 mentions)
Eosin y solution, by Fujifilm (1 mentions)
Eclipse ci, by Nikon (1 mentions)
Rm2245, by Leica (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Igf 1, by Abcam (1 mentions)
42 protocols using mayer s hematoxylin
1
Histological analysis of adipose tissue
2
Immunohistochemical Analysis of β-catenin and IGF-1
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Paraformaldehyde-fixed paraffin-embedded tissues were cut into 4-μm sections. Paraffin sections were deparaffinized and rehydrated. For antigen retrieval, the slides were autoclaved in 10 mM sodium citrate buffer. Endogenous peroxidase was blocked with 0.345% H2O2 for 30 min, and sections were further blocked with 5% BSA in PBS for 30 min. The sections were incubated overnight at 4°C with the following primary antibodies: β-catenin (1:100; BD transduction laboratory, CA, USA) and IGF-1 (1:100; Abcam, MA, USA). Sections were incubated with biotinylated anti-mouse secondary antibody (1:200) (Dako, Glostrup, Denmark) for 1 h at room temperature, followed by incubation with avidin–biotin complexes (Vector Laboratories, CA, USA). Staining was performed with 3,3′-diaminobenzidine (DAB) (Vector Laboratories) as explained in the manufacturer’s protocol. Counterstaining was performed with Mayer’s hematoxylin (Muto Pure Chemicals, Tokyo, Japan). The DAB-stained preparations were visualized with a TE-2000 general optical microscope (Nikon, Tokyo, Japan).
3
Immunohistological Analysis of Tumor Tissues
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Immunohistological experiments using surgically obtained tumor tissues from the patients were approved by the research ethics committee of the Niigata University (authorization number; #2015‐2334, 2016‐0089), and informed consent was obtained from all patients to perform the analyses. Immunohistochemistry was implemented using paraffin‐embedded section. Deparaffinization of paraffin‐embedded section was accompanied by antigen retrieval by using Pascal pressure chamber (Dako, USA) in sodium citrate buffer. Antigen‐retrieved sections were incubated with anti‐human Crb3a monoclonal (1 μg/mL, generated in our study) and/or anti‐FGFR1 (1:100 dilution, #sc‐121, Santa Cruz Biotechnology, USA) in PBS including 1.0% bovine serum albumin (#017‐25771, Wako Pure Chemical Industries), and stained using EnVision+ reagent (#K400011, Dako). DAB‐stained sections were counterstained with Mayer's hematoxylin (#30004, Muto Pure Chemicals, Japan). Fluorescent dye‐labeled secondary antibodies were employed for double staining.
4
Kidney in situ Hybridization Protocol
5
Visualizing Golli-MBP mRNA in Mouse Brain
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The ViewRNA ISH Tissue 1-Plex Assay is a valuable method for visualizing target mRNA localization in tissue sections using highly specific and branched DNA signal amplification technology (Vie-wRNA Tissue 1 Plex Assay Kit: #TFA-QVT0050, ViewRNA Tissue 1 plex Signal Amplification Kit: TFA-QVT0200, Thermo Fisher Scientific). Golli-MBP specific TYPE1 (Red signal) probe sets based on the sequence within 869-4,820 bases of NM_001025245.1 (Mus musculus MBP, transcript variant 8, mRNA) were produced by VERITAS Co., Ltd (Tokyo, Japan). Frozen brain sections, 12 µm in thickness, were obtained from 8-week-old C57BL6/J mice. These sections underwent pretreatment with a protease solution at 40 °C for 40 min, followed by hybridized with Golli-MBP specific probe sets at 40 °C for 3 h. Subsequently, signals were detected and amplified using the Fast Red substrate in accordance with the manufacturer's instructions. Following detection, the sections were counterstained with Mayer's hematoxylin (#30002, Muto Pure Chemicals Co., Ltd., Tokyo, Japan) and mounted with UltraMount Permanent Mounting Medium (#S1964, DAKO) to fix Fast Red signals.
6
Immunohistochemical Analysis of IGF-1 and Ki-67
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Formaldehyde-fixed tissues were embedded in paraffin and sectioned at 4 μm. All of the sections were subjected to H&E staining and immunohistochemical staining for IGF-1 and Ki-67. Quantitative analyses were performed by microscopically counting the numbers of Ki-67-positive cells per field. The antibodies used in this study are listed in Supplementary Table 2 . The sections were blocked with 1% hydrogen peroxide in 50% methanol before probing with primary antibodies. The antigens on the paraffin sections were then retrieved by autoclaving in 0.01 M citrate buffer (pH 6.0) for 10 min and visualized using a ChemMate Envision kit with a horseradish peroxidase/3,3′-diaminobenzidine kit. All sections were counterstained with Mayer's hematoxylin (Muto Pure Chemicals, Tokyo, Japan).
7
Silane-Coated Glass Slide Staining
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The sections on silane-coated glass slides were stained as follows: PBS for 10 s, Mayer’s hematoxylin (Muto Pure Chemicals) in aqueous solution for 30 s, DEPC-treated water for 5 s, eosin (Muto Pure Chemicals) in ethanolic solution for 30 s, followed by dehydration with 100% ethanol for 10 s, and Hemo-D (d-limonene; Falma, Tokyo, Japan) for 60 s.
8
Histological Analysis of Liver Tissue
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Liver specimens were fixed overnight in 4% paraformaldehyde in phosphate buffered saline (PBS) and replaced into 30% sucrose/PBS liquid. Samples were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Co.,Ltd., Tokyo), cut by 10 µm. For Sirius red staining, sections were washed in PBS for 5 min, counter stained with Mayer's Hematoxylin for 10 min, washed with running water for 2 min and subsequently soaked in hydrochloric acid alcohol (0.5% HCl in 70% EtOH) for 1 min. Sections were then stained with 0.03% Sirius red (Direct red 80, SIGMA-ALDRICH) in saturated picric acid solution for 15 min. HE staining was performed using Mayer's Hematoxylin (MUTO PURE CHEMICALS CO.,LTD., Tokyo) and Eosin (SIGMA-ALDRICH, St. Louis, USA).
9
Porcine Pancreatic Elastase and Methacholine Chloride Protocol
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Porcine pancreatic elastase (PPE) and methacholine chloride (methacholine) were obtained from Sigma-Aldrich (St. Louis, MO, United States). Novo-Heparin for injection was from Mochida Pharmaceutical, Co. (Tokyo, Japan). Chloral hydrate was from Nacalai Tesque (Kyoto, Japan). Diff-Quik was from Sysmex, Co. (Kobe, Japan). Formalin neutral buffer solution was from WAKO Pure Chemicals (Tokyo, Japan). Mayer’s hematoxylin, 1% eosin alcohol solution and malinol were from MUTO Pure Chemicals (Tokyo, Japan).
10
Histological Analysis of Submandibular Glands
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The SMGs from the respective mice were fixed with 4% (w/v) paraformaldehyde at 4°C overnight and equilibrated with 30% (w/v) sucrose. The specimens were frozen in OCT compound (Sakura Finetek Japan, Tokyo, Japan) and cut into 10-μm sections. For hematoxylin and eosin (HE) staining, the sections were stained with Mayer's Hematoxylin (Muto pure chemicals, Tokyo, Japan) and 1% Eosin Alcohol Solution (Muto pure chemicals) for 5 min each. For Azan staining, the sections were incubated with 5% (v/v) trichloroacetic acid and 5% (v/v) potassium dichromate for 10 min, Azocarmine G Solution (Wako Pure Chemical Industries, Osaka, Japan) for 30 min, 0.1% (v/v) aniline in 70% (v/v) ethanol for 3 sec, 0.1% (v/v) acetic acid in 95% (v/v) ethanol for 1 min, 5% (w/v) phosphotungstic acid for 60 min, and Aniline blueorange G solution (Wako Pure Chemical Industries) for 3 min. The slides were evaluated under the light microscope (BZ-X710; Keyence Corporation, Osaka, Japan).
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