Paraffin-embedded tissues were sliced at 4 µm, deparaffinized in xylene, and rehydrated in an ethanol gradient from 100% to 70%. Antigen retrieval was carried out in Tris-EDTA, pH 9.0 (preheated), before application of 1% H2O2 in methanol. After two washes in normal antibody diluent (Immunologic), the monoclonal antibody (MAb) mouse anti-IBV M protein 25.1 (Prionics, Lelystad, The Netherlands), cross-reacting with TCoV and GfCoV (5 (link)), was applied for 1 h at room temperature (RT). Slides were washed in phosphate-buffered saline (PBS)–0.1% Tween, and an EnVision kit (catalog no. K4001; Dako) was used for anti-mouse secondary antibody staining according to the manufacturer’s protocol. Slides were washed three times in PBS, and viral M protein presence was visualized with aminoethylcarbazole (AEC). The tissues were counterstained with hematoxylin and mounted with AquaMount (Merck).
Aquamount
Manufactured by Merck Group
Sourced in Germany
Aquamount is a water-soluble mounting medium used for microscopy applications. It is designed to prepare and preserve biological specimens for analysis under a microscope.
Automatically generated - may contain errors
Lab products found in correlation
Normal antibody diluent, by Immunologic (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
Diaminobenzidine dab substrate, by Agilent Technologies (1 mentions)
Anti β2, by Abcam (1 mentions)
Dfc480 camera, by Leica (1 mentions)
Pt link, by Agilent Technologies (1 mentions)
Dm4500b microscope, by Leica (1 mentions)
Autostainer, by Agilent Technologies (1 mentions)
N histofine kit, by Nichirei Biosciences (1 mentions)
Anti s100a4, by Abcam (1 mentions)
3 amino 9 ethyl carbazol, by Merck Group (1 mentions)
Avidin biotin peroxidase complex abc, by Vector Laboratories (1 mentions)
Biotinylated goat anti rabbit ab, by Vector Laboratories (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dfc480, by Leica (1 mentions)
Dm4500b, by Leica (1 mentions)
Bm purple ap substrate, by Roche (1 mentions)
7 protocols using aquamount
1
Immunohistochemical Detection of Coronavirus M Protein
2
Dual Immunohistochemical Analysis of Hematological Markers
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Double immuno‐enzymatic labelling of biopsies’ sections following the described pretreatment was carried out. Primary antibodies were incubated for 30 minutes at room temperature, and a diaminobenzidine (DAB) substrate (DakoCytomation) was then used for the detection of antibody binding. Sections were then incubated for 30 minutes with the second antibody. The second reaction was detected by means of a Vector Blue Alkaline Phosphatase or a Fast Red. The sections were washed in tap water and mounted in aquamount (Merck). Double immunostaining was carried out to analyse the expression of CD71, myeloperoxidase, CD61 and GATA‐1 in combination with CAL2 antibody.
The cases were reviewed by an expert haematopathologist (TM), a co‐author of this paper.
The cases were reviewed by an expert haematopathologist (TM), a co‐author of this paper.
3
Immunohistochemical Analysis of Tumor Biomarkers
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The spheroids were fixed in Carnoy fixative (Ethanol:Chloroform:Glacial acetic acid 6:3:1) and embedded in paraffin. Patient tumour tissues were fixed in neutralized formalin and embedded in paraffin. Antigen retrieval was performed at low pH retrieval buffer (EnVision® Flex Target Retrieval Solution Low pH) at PT-Link (Dako). Immunohistochemical analysis was performed on an automated immunostainer (Dako Autostainer) using DakoCytomation EnVision®+ System-HRP (DAB) according to the manufacturer’s instructions. For CA IX staining, the sections were labelled with M75 antibody (hybridoma medium) diluted 1:100 for 1 h at room temperature. For β1 and β2 adrenoreceptors and Ki-67 the sections were incubated with anti β1 (Invitrogen, Carlsbad, CA, USA, 1:500), anti β2 (Abcam, 1:250) overnight at 4 °C, for Ki-67 (DAKO, 1:100) for 1h at room temperature and, after washing, incubated with secondary anti mouse-HRP or anti rabbit-HRP antibody for 30 min at room temperature. Staining was visualized using 3,3’-diaminobenzidine (DAB) as a chromogenic substrate for 1 min. The sections were counterstained with Mayer’s haematoxylin and mounted in Aquamount (Merck, Darmstadt, Germany). The stained sections were examined using a Leica DM4500B microscope and images were captured by a Leica DFC480 camera.
4
Liver Immune Cell Profiling
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Double perforin/CD56 and perforin/CD3 immunostainings were performed on consecutive 4 µm thick liver sections (n = 4) from formalin-fixed, paraffin-embedded tissue, as described in [33] (link). Slides were first immunostained with the anti-perforin Ab2 antibody (Clone 5B10, Neomarkers) and revealed with the N-Histofine kit (Nichirei Biosciences Inc). Fast red was used as the chromogen for perforin immunodetection. Then, antigen retrieval was performed in Tris-citrate buffer for CD56 or citrate buffer for CD3. Slides were incubated with the primary anti-CD56 (clone 1B6, Novocastra) or anti-CD3 (polyclonal rabbit CD3, Dako) antibodies and then incubated with the secondary antibody using the Envision kit. DAB was used as the chromogen for CD56 or CD3 immunodetection. All sections were counterstained with Harris hematoxylin, washed in tap water and mounted in aquamount (Merck). Sur-fixation in glutaraldehyde (0.05% in PBS pH 8.6) followed each incubation with the primary or secondary antibody.
5
Immunohistochemical Labeling of Fibroblast-like Cells
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For labeling fibroblast-like cells, the polyclonal antibody antiS100a4 ab27957 (Abcam, Cambridge, UK) was used.
Transwell cultures were embedded after 10 and 21 days in Tissue-Tek (Sakura, Zoeterwoude, The Netherlands) and frozen at − 20 °C. Samples were cut into 10 µm serial-sections, mounted on superfrost glass slides (Menzel, Braunschweig, Germany), fixed in acetone, and dried at room temperature. After re-hydrating with 0.2% Tween (Merck, Darmstadt, Germany) in PBS sections were incubated for 1 h at RT with ABO-Serum (Biotest, Dreieich, Germany) mixed with horse serum (both 1:20 in PBS with 3% Bovine Serum Albumin (BSA; Sigma-Aldrich Co.) to block unspecific binding sites. Incubation with the first antibody (AB) anti-S100A4 (Abcam) (1:100 in AB dilution buffer (DCS Innovative, Hamburg, Germany)) for 1 h at RT was performed. After washing, incubation with the biotinylated goat anti-rabbit AB (Vector Laboratories, Burlingame, USA) (1:200 dilution with AB dilution solution) was performed for 30 min at RT. After washing, the sections were treated with avidin–biotin–peroxidase complex (ABC; Vector Laboratories) for 30 min at RT. Then, the sections were washed. Staining was developed using 3-Amino-9-Ethyl Carbazol (Sigma-Aldrich Co.) and counterstained with hemalaun. The sections were then embedded in Aquamount (Merck, Darmstadt, Germany).
Transwell cultures were embedded after 10 and 21 days in Tissue-Tek (Sakura, Zoeterwoude, The Netherlands) and frozen at − 20 °C. Samples were cut into 10 µm serial-sections, mounted on superfrost glass slides (Menzel, Braunschweig, Germany), fixed in acetone, and dried at room temperature. After re-hydrating with 0.2% Tween (Merck, Darmstadt, Germany) in PBS sections were incubated for 1 h at RT with ABO-Serum (Biotest, Dreieich, Germany) mixed with horse serum (both 1:20 in PBS with 3% Bovine Serum Albumin (BSA; Sigma-Aldrich Co.) to block unspecific binding sites. Incubation with the first antibody (AB) anti-S100A4 (Abcam) (1:100 in AB dilution buffer (DCS Innovative, Hamburg, Germany)) for 1 h at RT was performed. After washing, incubation with the biotinylated goat anti-rabbit AB (Vector Laboratories, Burlingame, USA) (1:200 dilution with AB dilution solution) was performed for 30 min at RT. After washing, the sections were treated with avidin–biotin–peroxidase complex (ABC; Vector Laboratories) for 30 min at RT. Then, the sections were washed. Staining was developed using 3-Amino-9-Ethyl Carbazol (Sigma-Aldrich Co.) and counterstained with hemalaun. The sections were then embedded in Aquamount (Merck, Darmstadt, Germany).
6
Immunohistochemical Analysis of Melanin-Rich Tissues
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Dissected tumours were formalin-fixed and embedded in paraffin according to the standard histological procedure. Because of melanin presence in tissues, sections were bleached using potassium permanganate solution (2 g/l) for 20 min at 65 °C. After rinsing in PBS, sections were immersed in 1% oxalic acid for 1 min at room temperature followed by rinsing in PBS for 10 min. Immunohistochemistry was performed on Dako Autostainer employing DakoCytomation EnVision®+ System-HRP (DAB) for use with Mouse Primary Antibodies according to the manufacturer’s instructions. The sections were labelled with M75 antibody in hybridoma medium diluted 1:100 for 1 h at room temperature and after washing incubated with secondary anti-mouse-HRP antibody for 30 min at room temperature. Absence of nonspecific binding of the secondary antibody was confirmed on parallel sections by omitting the primary antibody. Staining was visualised using 3,3′-diaminobenzidine (DAB) as a chromogenic substrate for 1 min. The sections were counterstained with Mayer’s haematoxylin and mounted in Aquamount (Merck Millipore, MA, USA). The stained sections were examined using a Leica DM4500B microscope and images were captured by a Leica DFC480 camera.
7
In Situ Hybridization of miR-207 in Cochlea
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ISH was performed to confirm the expression of miR-207 in cochlea hair cells in vivo. Cochleas of C57BL/6 mice were carefully and quickly resected, followed by removing the stapes from the oval window and opening the cochlea apex. We then placed the cochleas in fresh 4% paraformaldehyde (PFA) in PBS overnight at 4 °C. After decalcification with 0.1 M ethylenediaminetetraacetic for 3 days at 4 °C, the cochleas were embedded in paraffin and sectioned at 5 μm. ISH was performed as described.32 (link) Briefly, sections were hybridized with labeled LNA probe, which was detected using AP-conjugated sheep anti-DIG Fab fragment and BM Purple AP Substrate (Roche). The sections were then fixed by 4% PFA for 20 min finish with milliQ water (Millipore, Billerica, MA, USA) rinses. Slides were coverslipped with Aquamount (Merck, Darmstadt, Germany). The slides were observed using a light microscope.
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