Immunohistochemistry was performed on ovaries (3/group) collected 14 hr after mating, fixed in 10% formaldehyde for 24 hr, processed and paraffin embedded. Rehydrated sections of 5 µm were incubated in 0.3% hydrogen peroxide in TBS to block endogenous peroxidase activity and then in primary antibody solution (goat polyclonal anti-human ZP4 (Santa-Cruz sc-49586) dilution 1:2000 in TBS-1% BSA solution) for 1 hr at room temperature in a moist chamber. After a brief wash in TBS, sections were incubated in secondary antibody solution (ImmPRESS horse anti-goat IgG HRP, Vector Labs MP-7405) for 30 min at 37°C and immunoreaction was revealed with 3–3´diaminobencidine. Finally, sections were counterstained with Harry´s hematoxylin (Thermo Scientific), dehydrated, cleared and mounted. Positive immunoreaction was identified as a dark-brown precipitated. The images were collected with a Leica DM 6000 microscope and Software Leica Application Suite.
Mp 7405
Manufactured by Vector Laboratories
The MP-7405 is a high-performance microplate reader designed for a variety of laboratory applications. It features a compact and user-friendly design, with a range of capabilities to support your research needs.
Automatically generated - may contain errors
Lab products found in correlation
Mp 7402, by Vector Laboratories (3 mentions)
Sk 4103 100, by Merck Group (2 mentions)
Apotome microscope, by 3DHISTECH (2 mentions)
Axiocam mrm camera, by 3DHISTECH (2 mentions)
Mp 7401, by Vector Laboratories (2 mentions)
Ghs132, by Merck Group (2 mentions)
Prolong gold, by Thermo Fisher Scientific (2 mentions)
Pannoramic midi 2, by 3DHISTECH (2 mentions)
Leica dm6000 microscope, by Leica camera (1 mentions)
Hematoxylin, by Leica Biosystems (1 mentions)
Cleaved caspase 3 asp175 5a1e, by Cell Signaling Technology (1 mentions)
Mp 7500, by Vector Laboratories (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Sk 4100, by Vector Laboratories (1 mentions)
Bromodeoxyuridine (brdu), by Roche (1 mentions)
Rnascope vs universal ap assay, by Advanced Cell Diagnostics (1 mentions)
Discovery ultra automated stainer, by Roche (1 mentions)
Sars cov 2 specific probe, by Advanced Cell Diagnostics (1 mentions)
Af933, by R&D Systems (1 mentions)
Chromomap dab kit, by Roche (1 mentions)
6 protocols using mp 7405
1
Immunohistochemical Analysis of ZP4 in Ovaries
2
Immunohistochemistry of FFPE Tissues
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IHC on formalin-fixed, paraffin-embedded tissue was performed as previously described.20 (link),50 (link) Five-micron tissue sections were cut and transferred onto Superfrost-Plus slides (Fisher Scientific). Tissue sections were deparaffinized, rehydrated and antigen retrieval was performed by microwaving in 10 mM citrate buffer, pH 6.0 for 10 minutes. Sections were incubated with 3% H2O2 for 15 minutes to quench endogenous peroxidase activity. Tissue sections were incubated with primary antibody (diluted in 3% skim milk, 1% BSA in PBS) against human CAIX (1:200, M75, Bioscience), BrdU (1:10, Roche), cleaved caspase 3 (Asp175) 5A1E (1:100, Cell Signaling), or pimonidazole (1:500) for overnight at 4°C. ImmPRESS species specific HRP secondary (MP-7405, MP-7500) and DAB peroxidase HRP Substrate (SK-4100) were used according to the manufacturer’s instructions (Vector Laboratories). Tissues were counterstained with hematoxylin (Leica Biosystems). For quantification, at least 5 randomly selected fields of view (FOV) at 20x magnification were imaged from 1 section/tumor and 5-6 tumors were analyzed/group. The number of positive cells in each image was counted or the percent area of positive staining was quantified using ImageJ (v1.48, National Institutes of Health, USA).
3
SARS-CoV-2 Detection in Tissue Samples
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Tissues were fixed in 10% neutral buffered formalin for a minimum of 7 days with 2 changes according to IBC-approved standard operating procedure. Tissues were processed with a Sakura VIP-6 Tissue Tek, on a 12-hour automated schedule, using a graded series of ethanol, xylene, and PureAffin. Embedded tissues were sectioned at 5 μm and dried overnight at 42 °C prior to staining with hematoxylin and eosin. Chromogenic detection of SARS-CoV-2 viral RNA was performed using the RNAscope VS Universal AP assay (Advanced Cell Diagnostics Inc.) on the Ventana Discovery ULTRA stainer using a SARS-CoV-2 specific probe (Advanced Cell Diagnostics Inc. cat. 848569). In situ hybridization was performed according to the manufacturer’s instructions. ACE2 immunoreactivity was detected using R&D Systems AF933 at a 1:100 dilution. The secondary antibody is the Vector Laboratories catalog number MP-7405 ImmPress anti-goat polymer. The tissues were then processed for immunohistochemistry using the Discovery Ultra automated stainer (Ventana Medical Systems) with a ChromoMap DAB kit (Roche Tissue Diagnostics cat. 760–159).
4
Nerve and Hair Cell Staining Protocol
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For staining of nerve terminals, we used paraffin sections (10 µm). Primary antibodies against class III beta tubulin (TuJ1 MMS-435P, Lot #D13AF00117, Covance, 1:500; Lee et al., 1990 (link)) were incubated in 2.5% normal horse serum (NHS) in PBS overnight at room temperature. After three washes in PBS the secondary antibodies (MP-7402, Vector Laboratories, Burlingame, CA, USA) were incubated for 1 h at room temperature. Following an additional three PBS washes the staining was visualized with the chromogen 3,3′-diaminobenzidine (SK-4105, Vector Laboratories). Nuclear Fast Red (C.I. 60760; Carl Roth, Karlsruhe, Germany) served as a counterstain. Hair cells were stained on cryo-sections (12 µm). After a heat-mediated antigen retrieval (H-3301, Vector Laboratories) the sections were washed three times in PBS and primary antibodies against otoferlin (Sc-50159, C-15, Lot #A2930; Santa Cruz, 1:1000) were incubated in 2% milk, 0.1% Triton in PBS overnight at room temperature (Lauwers et al., 2013 (link)). After three washes in PBS the secondary antibodies (MP-7405, Vector Laboratories) were incubated for 1 h at room temperature. Staining was than visualized with DAB as described above.
5
Adrenal Tissue Immunohistochemistry Protocol
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Adrenals were fixed in 10% normal buffered formalin (Fisher Scientific, 23-427098) for 24 hours at room temperature, paraffin-embedded, and cut into 5µm sections. Antibody information and staining conditions are listed in Supplementary Table 2. Endogenous peroxidase activity was blocked with 0.3% H202 for 30 minutes at room temperature. For brightfield IHC, primary antibodies were detected with HRP polymer solution (Vector Laboratories, MP-7401 (anti-rabbit), MP-7402 (anti-mouse), or MP-7405 (anti-goat)) and DAB EqV peroxidase substrate (Vector Laboratories, SK-4103-100), nuclei were counterstained with hematoxylin (Sigma, GHS132), and slides were mounted using Vectamount (Vector Laboratories, H-5000-60). For immunofluorescence (IF), primary antibodies were detected with HRP polymer solution and Alexa fluor tyramide reagent (Thermo Fisher, B40953 (488), B40955 (555), or B40958 (647)), nuclei were counterstained with Hoechst (Thermo Fisher, H3570), and slides were mounted using ProLong Gold (Thermo Fisher, P36930). Imaging was performed on a Zeiss Apotome microscope with an AxioCam MRm camera or a Pannoramic MIDI II (3DHISTECH) digital slide scanner. Quantification was performed using QuPath digital pathology software (Version 0.3.0).
6
Adrenal Tissue Immunohistochemistry Protocol
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Adrenals were fixed in 10% normal buffered formalin (Fisher Scientific, 23-427098) for 24 hours at room temperature, paraffin-embedded, and cut into 5µm sections. Antibody information and staining conditions are listed in Supplementary Table 2. Endogenous peroxidase activity was blocked with 0.3% H202 for 30 minutes at room temperature. For brightfield IHC, primary antibodies were detected with HRP polymer solution (Vector Laboratories, MP-7401 (anti-rabbit), MP-7402 (anti-mouse), or MP-7405 (anti-goat)) and DAB EqV peroxidase substrate (Vector Laboratories, SK-4103-100), nuclei were counterstained with hematoxylin (Sigma, GHS132), and slides were mounted using Vectamount (Vector Laboratories, H-5000-60). For immunofluorescence (IF), primary antibodies were detected with HRP polymer solution and Alexa fluor tyramide reagent (Thermo Fisher, B40953 (488), B40955 (555), or B40958 (647)), nuclei were counterstained with Hoechst (Thermo Fisher, H3570), and slides were mounted using ProLong Gold (Thermo Fisher, P36930). Imaging was performed on a Zeiss Apotome microscope with an AxioCam MRm camera or a Pannoramic MIDI II (3DHISTECH) digital slide scanner. Quantification was performed using QuPath digital pathology software (Version 0.3.0).
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