Immunohistochemistry was performed as described previously [62 (link)] with some modifications. Paraffin sections of the mouse prostate were deparaffinized and underwent epitope unmasking using a heat antigen retrieval step. Section-staining was performed using the manufacturer’s protocol for Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer (Abcam ab236466). Antibodies used were: rabbit anti-CD45 (Abcam ab208022, 1:1000), anti-CD3 (Abcam ab5690, 1:300), and anti-CD19 (Cell Signaling 90176, 1:800). Hematoxylin and eosin (H&E) staining was performed by UCLA’s Translational Pathology Core Laboratories. Entire areas of an H&E-stained prostate section were captured by Axio Imager M2 (Zeiss) from two separate sections from a prostate, from four animals per age group. The sectional areas of the tertiary lymphoid structures was calculated using Zen 3.5 software (Zeiss).
Mouse and rabbit specific hrp dab ihc detection kit micro polymer
Manufactured by Abcam
Sourced in United Kingdom
The Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer is a laboratory equipment used for immunohistochemistry (IHC) applications. It contains a micro-polymer-based detection system that specifically recognizes and binds to mouse and rabbit primary antibodies, enabling the visualization of target antigens in tissue sections.
Automatically generated - may contain errors
Lab products found in correlation
Sku h 3300 250, by Vector Laboratories (2 mentions)
Axio imager m2, by Zeiss (1 mentions)
Zen 3, by Zeiss (1 mentions)
H 5000, by Vector Laboratories (1 mentions)
Vectamount, by Vector Laboratories (1 mentions)
Hematoxylin qs h 3404, by Vector Laboratories (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Permount mounting medium, by Thermo Fisher Scientific (1 mentions)
Dm400b, by Leica (1 mentions)
Anti tnf α antibody, by Abcam (1 mentions)
Anti il 8 antibody, by Abcam (1 mentions)
Proteinase k, by Abcam (1 mentions)
Anti il 6 antibody, by Abcam (1 mentions)
6 protocols using mouse and rabbit specific hrp dab ihc detection kit micro polymer
1
Prostate Immune Cell Quantification
2
Quantitative CDK4/6 Immunohistochemistry
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Immunohistochemistry staining analysis was performed by using Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer (Abcam) and anti-CDK4 and CDK6 antibodies. (B) The nucleus staining analysis was performed by counting DAB-positive nucleus staining in random nine IHC images.
3
Immunohistochemical Staining of Tissue Slides
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Tissue slides were baked for 1 hour at 65°C, deparaffinized in xylene, and rehydrated through a series of graded alcohols. After diH2O washes, slides were treated with antigen unmasking solution (1:100; SKU H-3300-250, Vector Laboratories) at 95°C for 12 min according to the manufacturer’s protocol. Hydrogen peroxide, blocking, primary antibody binding, HRP-conjugated secondary antibody, and 3, 3′-diaminobenzidine (DAB) were done using the Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer (ab236466, Abcam). Before the protein block, tissues were treated with 0.1% Triton X-100 diluted in 1× tris-buffered saline (TBS) for 5 min to unmask the antigens expressed in the nucleus. After overnight primary antibody incubation at 4°C and endogenous treatment with secondary anti-rabbit antibody at room temperature (RT) for 20 min, the staining was visualized with DAB with exposure times synchronized throughout all tissue samples within an antibody group for the exact same time. All slides were counterstained with hematoxylin (Hematoxylin QS, H-3404, Vector Laboratories) for 20 s at RT, dehydrated in ethanol, cleared in xylene, and mounted with VectaMount (permanent mounting medium, H-5000, Vector Laboratories).
4
Immunohistochemical Staining of Tissue Slides
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Tissue slides were baked for 1 hour at 65°C, deparaffinized in xylene, and rehydrated through a series of graded alcohols. After diH2O washes, slides were treated with antigen unmasking solution (1:100; SKU H-3300-250, Vector Laboratories) at 95°C for 12 min according to the manufacturer’s protocol. Hydrogen peroxide, blocking, primary antibody binding, and secondary antibody binding were done using the Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer (ab236466, Abcam). Before the protein block, tissues were treated with 0.1% Triton X-100 diluted in 1× TBS for 5 min to unmask the antigens expressed in the nucleus. After overnight, primary antibody incubation at 4°C and endogenous treatment with secondary fluorescent antibodies at RT for 1 hour. All slides are mounted with ProLong Gold antifade reagent with DAPI (P36935, Invitrogen).
5
Immunohistochemical Analysis of 8-oxo-dG
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We used sorted, fixed, embedded and sectioned ISCs for 8-oxo-dG staining, a known marker of oxidative DNA damage. Sections were deparaffinized, sequentially rehydrated, and treated with a primary antibody specific to 8-oxo-dG (Trevigen, Gaithersburg, MD) according to a protocol described previously [15 (link)]. Signals were detected using DAB substrate provided in the Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer (Cat# ab236466, Abcam, Cambridge, MA) according to the manufacturer’s instruction. Sections were counterstained with hematoxylin, sequentially dehydrated, and mounted using Permount mounting medium (Thermo Fisher Scientific). Mounted slides were visualized under a bright field microscope and images were captured. Images were quantified using ImageJ v1.51 and the error bar represents SEM.
6
Immunohistochemical Staining of Inflammatory Markers
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Immunohistochemical staining was performed according to the manufacturer’s procedure with the Mouse and Rabbit Specific HRP/DAB IHC Detection Kit-Micro-polymer (Cat No. ab236466; Abcam, UK). Proteinase K (Cat No. ab64220; Abcam, UK) was used for antigen retrieval. Sections taken from paraffin blocks with adhesive were submerged in 3% H2O2 peroxidase block solution and then the protein block solution was poured out. The sections were exposed to 1:100 anti-TNF-α antibody (Cat No. ab6671; Abcam, USA), anti-IL-6 antibody (Cat No. ab6672; Abcam, USA), and anti-IL-8 antibody (Cat No. ab34100; Abcam, USA) and were left at room temperature for 1 h. Subsequently, Mouse Identification Reagent (Complementary) solution was added to the slides and they were exposed to goat anti-rabbit HRP-conjugate. Slides were stained with DAB (3,3′-diaminobenzidine tetrahydrochloride). After counterstaining with Mayer’s hematoxylin, slides were sealed with coverslips and evaluated under a light microscope (Leica DM 400B, Leica, Germany). Negative control slides were also stained according to the same procedure and PBS was used instead of primary antibodies. Immunohistochemical staining was scored semiquantitatively as revealing low (+), moderate (++), or high (+++) expression.
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