For immunohistochemistry (IHC), tissue sections of monkey eyes were de-paraffined with xylene and rehydrated through an ethanol series and phosphate-buffered saline (PBS). Antigen retrieval was performed by microwave treatment with ethylenediaminetetraacetic acid (EDTA)/Tris buffer, pH9. Endogenous peroxidase was blocked with 0.3% H2O2 in methanol for 30 minutes, followed by incubation with Protein Block (Dako Pathology Solutions, Agilent, Santa Clara, CA) and avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA). The sections were incubated with anti-CD31 mouse monoclonal antibody (Dako Pathology Solutions) at 4°C overnight, then incubated with biotin-conjugated rabbit anti-mouse Ig (Dako Pathology Solutions), for 30 minutes at room temperature followed by the addition of peroxidase conjugated streptavidin (Nichirei, Tokyo, Japan) for 5 minutes. Peroxidase activity was visualized by diaminobenzidine. The sections were counterstained with Mayer's Hematoxylin Solution (Muto Pure Chemicals, Tokyo, Japan), dehydrated, and then mounted with Malinol (Muto Pure Chemicals).
Mayer s hematoxylin solution
Manufactured by Muto Pure Chemicals
Sourced in Japan
Mayer's hematoxylin solution is a laboratory reagent used for the staining of biological samples. It is a component of the hematoxylin and eosin (H&E) staining method, which is a widely used technique in histology and pathology for the visualization of cellular structures.
Automatically generated - may contain errors
Lab products found in correlation
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Peroxidase conjugated streptavidin, by Nichirei Biosciences (1 mentions)
Malinol, by Muto Pure Chemicals (1 mentions)
β actin 1 19, by Santa Cruz Biotechnology (1 mentions)
Recombinant human tumor necrosis factor α, by Thermo Fisher Scientific (1 mentions)
Egmtm 2 endothelial cell growth medium 2 bulletkittm, by Lonza (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ez ldh cytotoxicity assay kit, by DoGenBio (1 mentions)
Ez cytox cell viability assay kit, by DoGenBio (1 mentions)
Ab16731, by Abcam (1 mentions)
Ab22604, by Abcam (1 mentions)
Eosin y solution, by Fujifilm (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Mayer s hematoxylin solution, by Fujifilm (1 mentions)
New eosin solution type m, by Muto Pure Chemicals (1 mentions)
Bouin s fluid, by Polysciences (1 mentions)
Microm hm 355, by Thermo Fisher Scientific (1 mentions)
2 isopropylmalic acid, by Merck Group (1 mentions)
Eosin y solution, by Muto Pure Chemicals (1 mentions)
7 protocols using mayer s hematoxylin solution
1
Immunohistochemical Staining of CD31 in Monkey Eyes
2
Endothelial Cell Signaling Pathway Analysis
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Antibodies against VEGF (A-20), MMP-9 (H-129), and β-actin (I-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In addition, antibodies against PI3K Class III (D4E2), phospho-PI3K Class III (Ser 249), AKT (5G3), phospho-AKT (Ser473), p38 (D13E1), phospho-p38 Thr180/Tyr182 (D3F9), ERK (p44/42), phospho-ERK (Thr202/Tyr204), phospho-p65 (Ser536) (93H1), p65 (C22B4), phospho-JNK (Thr183/Tyr185), JNK, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM was purchased from Lonza (Walkersville, MD, USA). The EZ-Cytox cell viability assay kit and EZ-LDH cytotoxicity assay kit were purchased from DoGenbio (Seoul, Korea). Recombinant human tumor necrosis factor (TNF)-α was purchased from Peprotech (Rocky Hill, NJ, USA). Mayer’s hematoxylin solution was purchased from Muto Pure Chemicals (Tokyo, Japan).
3
Immunohistochemical Analysis of Catalase and Glutathione Peroxidase in Tumor Tissues
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Sections of paraffin-embedded tumor tissues of the six treatment groups at days 1, 7, and 55 after treatment were deparaffinized and stained using the peroxidase-anti-peroxidase (PAP) IHC method (Dako REAL peroxidase blocking solution S2023, Glostrup, Denmark) with an anticatalase antibody (1:50, Abcam, ab16731) and antiglutathione peroxidase antibody (1:100, Abcam, ab22604). Nuclei were counterstained using the hematoxylin stain (Mayer’s hematoxylin solution, Muto Pure Chemicals Co., Tokyo, Japan).
4
Histological Analysis of Mouse Maxillae
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Maxillae from the mice were fixed in 4% paraformaldehyde (Wako Pure Chemical Industries Ltd., Osaka, Japan) overnight at 4°C and decalcified in decalcify solution B (Wako Pure Chemical Industries Ltd.) for 1 week. After decalcification, periodontal tissues were embedded in paraffin and sectioned at 5 μm in a mesio-distal orientation using a LEICA RA2245 microtome (Leica Microsystems, Wetzlar, Germany). Hematoxylin—eosin staining was performed using Mayer’s hematoxylin solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) and 1% Eosin Y solution (Wako Pure Chemical Industries Ltd.).
5
Histological Analysis of Male Mouse Reproductive Organs
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Testes, epididymides, and seminal vesicles from male mice (19–34 weeks old) were fixed in Bouin’s fluid (Polysciences, Warrington, PA, USA) at 4°C overnight. The fixed tissues were
dehydrated by increasing the ethanol concentration and then embedded in paraffin. The embedded tissues were sliced at 5- or 6-µm thickness using a Microm HM355 (Thermo
Fisher Scientific). For epididymides and seminal vesicles, sliced sections were stained with Mayer’s hematoxylin solution (Muto Pure Chemicals, Tokyo, Japan) for 3 min and counterstained
with New eosin solution Type M (Muto Pure Chemicals) for 3 min. For testes, the sliced sections were stained with 1% periodic acid solution (PAS, Wako) for 10 min, followed by treatment with
Schiff’s reagent (Wako) for 20 min, and then Mayer’s hematoxylin solution for 5 min. After dehydration with ethanol, the slides were mounted using Multi Mount 480 (Matsunami Glass, Osaka,
Japan). Spermatozoa from the cauda epididymis were dispersed in PBS, and the sperm morphology was observed using a differential interference contrast microscopy.
dehydrated by increasing the ethanol concentration and then embedded in paraffin. The embedded tissues were sliced at 5- or 6-µm thickness using a Microm HM355 (Thermo
Fisher Scientific). For epididymides and seminal vesicles, sliced sections were stained with Mayer’s hematoxylin solution (Muto Pure Chemicals, Tokyo, Japan) for 3 min and counterstained
with New eosin solution Type M (Muto Pure Chemicals) for 3 min. For testes, the sliced sections were stained with 1% periodic acid solution (PAS, Wako) for 10 min, followed by treatment with
Schiff’s reagent (Wako) for 20 min, and then Mayer’s hematoxylin solution for 5 min. After dehydration with ethanol, the slides were mounted using Multi Mount 480 (Matsunami Glass, Osaka,
Japan). Spermatozoa from the cauda epididymis were dispersed in PBS, and the sperm morphology was observed using a differential interference contrast microscopy.
6
Histochemical Liver Analysis Protocol
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Haematoxylin-eosin (HE) staining was performed with Mayer's Hematoxylin solution (Muto Pure Chemicals Co., Ltd., Tokyo, Japan) and Eosin Y solution (1% Eosin Y in 70% ethanol). For the analysis of lipid accumulation, the liver area containing lipid droplets was quantified morphometrically, in random 20× magnification fields of HE-stained sections, using ImageJ software.21 For the analysis of inflammation score, inflammatory cell infiltration levels were scored using the procedure described by Brunt et al. 22 The number of lobular inflamed foci was counted and scored (score 1 for <2 foci, score 2 for 2-4 foci, score 3 for >4 foci) using random 20× magnification fields of HE-stained sections.
For picrosirius red staining, the sections were incubated with 0.1% Sirius red (Direct Red 80; Sigma-Aldrich, St. Louis, MO) in Van Gieson Solution A (Muto Pure Chemicals Co., Ltd.) for 1 hour and rinsed with 0.01 N HCl. 23 The areas of the stained region were quantified in random 10× magnification fields of picrosirius red-stained sections using ImageJ software.
For picrosirius red staining, the sections were incubated with 0.1% Sirius red (Direct Red 80; Sigma-Aldrich, St. Louis, MO) in Van Gieson Solution A (Muto Pure Chemicals Co., Ltd.) for 1 hour and rinsed with 0.01 N HCl. 23 The areas of the stained region were quantified in random 10× magnification fields of picrosirius red-stained sections using ImageJ software.
7
Synthesis of Racemic-ACA Compound
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Racemic-ACA was supplied by Dr. H. Azuma, Graduate School of Engineering of Osaka City University. It was prepared according to the previously reported method [19] as following. Starting from 4-, 3-, and 2-hydroxybenzaldehyde, racemic-ACA was synthesized via a Grignard coupling reaction with vinylmagnesium bromide and further acetylation of the produced hydroxyl intermediate.
Mayer's hematoxylin solution and 1% eosin Y solution were purchased from Muto Pure Chemicals Co., LTD. (Tokyo, Japan). 2-Isopropylmalic acid was obtained from Sigma-Aldrich, Tokyo, Japan. Other chemicals used in this study were special-grade commercial products purchased from WAKO Pure Chemical Industries, Ltd. (Osaka, Japan).
Mayer's hematoxylin solution and 1% eosin Y solution were purchased from Muto Pure Chemicals Co., LTD. (Tokyo, Japan). 2-Isopropylmalic acid was obtained from Sigma-Aldrich, Tokyo, Japan. Other chemicals used in this study were special-grade commercial products purchased from WAKO Pure Chemical Industries, Ltd. (Osaka, Japan).
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