The paraffin section was placed in a 68 °C oven for 1 h, and washed three times with xylene for 7 min each for paraffin removal, then three times with 100% ethanol for 5 min each for xylene removal. The section was then rinsed with 90%, 80%, and 70% ethanol in this order, for 5 min each. The rinsed section was washed with PBS three times for 5 min each to remove alcohol completely. Next, the Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC Kit (Abcam Inc., Cambridge, UK) was used. Subsequently, the production of osteocalcin was monitored using the Slide Scanner (Axio Scan.Z1, Germany).
Mouse and rabbit specific hrp dab abc detection ihc kit
Manufactured by Abcam
Sourced in United Kingdom
The Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit is a labeling system used for immunohistochemistry (IHC) applications. It provides a sensitive detection method for visualizing target antigens in tissue sections or cells. The kit utilizes a biotin-avidin-horseradish peroxidase (HRP) amplification system and 3,3'-diaminobenzidine (DAB) as the chromogenic substrate to produce a colored reaction product.
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Lab products found in correlation
Cryostat, by Thermo Fisher Scientific (2 mentions)
Hematoxylin, by Merck Group (2 mentions)
Nanozoomer s60 slide scanner, by Hamamatsu Photonics (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
α actin, by Agilent Technologies (1 mentions)
Ndrg2, by Santa Cruz Biotechnology (1 mentions)
Histomount, by Thermo Fisher Scientific (1 mentions)
Axiocam mrc digital camera, by Zeiss (1 mentions)
Axio scope a1, by Zeiss (1 mentions)
Vecta elite kit, by Vector Laboratories (1 mentions)
Mounting medium, by Abcam (1 mentions)
Protein block solution, by Abcam (1 mentions)
Hematoxylin solution, by Abcam (1 mentions)
Signalstain antibody diluent, by Cell Signaling Technology (1 mentions)
Bx60 compound microscope, by Olympus (1 mentions)
Ab185430, by Abcam (1 mentions)
Tnf α, by Abcam (1 mentions)
Adamts5, by Abcam (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Pronase, by Roche (1 mentions)
18 protocols using mouse and rabbit specific hrp dab abc detection ihc kit
1
Immunohistochemical Analysis of Osteocalcin
2
Immunohistochemical Quantification of Ki67
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The expression of Ki67 in tumor tissues from nude mice was analyzed by immunohistochemical analysis. Briefly, the tissues were fixed with 4% formaldehyde for 24 h, embedded and cut into 4-μm-thick section. The sections were treated with 10 mmol/l sodium citrate buffer and incubated with anti-Ki67 antibody (1: 200 dilution) overnight at 4 °C. The positive signaling was stained by using a Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit (Abcam Trading (Shanghai) Company Ltd., Shanghai, China), and counterstained with hematoxylin. The relative integral optical density (IOD) of positive signaling was obtained by ImageJ software.
3
Tissue Microarray Immunohistochemistry Protocol
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Tissue microarray (TMA) slides were generously provided by Prof. Zhang Zhigang (Shanghai Cancer Institute, China). After deparaffinization and hydration in Milli-Q water, antigens were retrieved by boiling TMA slides in citrate buffer for 20 min. Then, endogenous peroxidase activity and nonspecific binding activity were blocked before incubation with the primary antibody at the recommended dilution (4 °C, overnight) in a wet box. Finally, immunoreactivity was visualized using the Mouse and Rabbit specific HRP/DAB (ABC) Detection IHC Kit (Abcam, Cambridge, MA, USA) in accordance with the manufacturer's instructions. Nuclei were counterstained with hematoxylin (Sigma-Aldrich). Images were taken by a NanoZoomer S60 slide scanner (Hamamatsu Photonics, Shizuoka, Japan) at a magnification of 20×. The expression levels of corresponding targets were determined by quantitative analysis. IHC scores were graded according to the percentage of stained cells as previously described 38 (link). Briefly, the scoring was assigned as follows: the percentage scores: 0, <5% of positively stained cells; 1, 5-50%; 2, 51-100%; the intensity scores: 0, absent or faint; 1, weak; 2, moderate; 3, strong.
4
Immunohistochemical and Immunofluorescence Staining Protocols
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Immunohistochemical staining was performed as described in our previous work (Li et al., 2019 (link)), using the Mouse- and Rabbit-specific HRP/DAB (ABC) Detection IHC Kit (ab64264; Abcam, Cambridge, United Kingdom) following the manufacturer’s protocol. Briefly, the tissue sections were deparaffinized and rehydrated. Epitope retrieval was performed in ethylenediaminetetraacetic acid (EDTA). After incubation with primary antibody overnight, HRP conjugated secondary antibody was used. For immunohistochemical detection, tissue was subsequently counterstained with diaminobenzidine, hematoxylin and hydrated. It is replaced the primary antibody with PBS as negative controls. Staining intensity was evaluated by ImageJ-Pro Plus 6.0 software. Pictures were captured under a Leica DMi8 microscope (Wetzlar, Germany).
Immunofluorescence staining of tissues was performed as described previously (Zhang et al., 2018 (link)).
Immunofluorescence staining of tissues was performed as described previously (Zhang et al., 2018 (link)).
5
Histological Analysis of Calcified Aortic Valve
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Three consecutive sections from the AS valve analyzed by MALDI-IMS were used for the histological analysis. These sections were stained using Verhoeff-Van Giesson staining protocol for elastic fibers and oil red for lipids.
Besides the consecutive sections, we used 4 different AS valves, selecting 4 sections with increasing level of calcification of each one, for IHC analyses. In this case, sections were blocked with 10% BSA in PBS Buffer with 0.1% Tween 20 and incubated for 1 h with the primary antibodies at room temperature. Specifically, these analyses were performed with antibodies against CD68, α-actin (DAKO), NDRG2 and collagen α3 Type VI (Santa Cruz). Secondary antibody conjugated with biotin, streptavidin-peroxidase and DAB was used for all immunostainings (Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit, Abcam). Besides, additionally sections from 4 stenotic valves were used to study the distribution of NDRG2 and collagen α3 Type VI along the different stages of the lesion. For an impartial analysis of the DAB staining, an orthonormal transformation of the RGB images by using an ImageJ plugin based on Ruifrok and Johnston’s method for color deconvolution was performed37 (link).
Besides the consecutive sections, we used 4 different AS valves, selecting 4 sections with increasing level of calcification of each one, for IHC analyses. In this case, sections were blocked with 10% BSA in PBS Buffer with 0.1% Tween 20 and incubated for 1 h with the primary antibodies at room temperature. Specifically, these analyses were performed with antibodies against CD68, α-actin (DAKO), NDRG2 and collagen α3 Type VI (Santa Cruz). Secondary antibody conjugated with biotin, streptavidin-peroxidase and DAB was used for all immunostainings (Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit, Abcam). Besides, additionally sections from 4 stenotic valves were used to study the distribution of NDRG2 and collagen α3 Type VI along the different stages of the lesion. For an impartial analysis of the DAB staining, an orthonormal transformation of the RGB images by using an ImageJ plugin based on Ruifrok and Johnston’s method for color deconvolution was performed37 (link).
6
Immunohistochemical Protein Localization in EBs
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In the deparaffinised sections of the EBs, the localisation of analysed proteins was revealed using the following antibodies: mouse anti-Sox17, mouse anti-CD184, mouse anti-integrin β1, rabbit anti-integrin α6 and mouse anti-E-cadherin. The sections were processed with Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit (ab64264; Abcam) in accordance with the manufacturer's protocol. The sections were embedded into Histomount (Life Technologies). The images of cross-sections were analysed using Cell^B image acquisition software.
7
Immunohistochemical Analysis of Tumor Proliferation
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Immunohistochemical analysis was performed on a thick (5 μm), paraformaldehyde-fixed, paraffin-embedded tumor tissue sections using the Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC Kit (Abcam Inc., Cambridge, United Kingdom) as reported precedingly (Wang et al., 2012 (link)). Briefly, tissue sections were immunostained with an anti-Ki-67 (1:50) or anti-PCNA (dilution 1:200). The number of Ki-67 or PCNA positive cells was determined in 10 randomly selected microscopic fields at ×400 magnification. The proliferation index of Ki-67 or PCNA was calculated as: the number of Ki-67 or PCNA positive cells/the total cell count × 100%. Immunofluore analyses for Ki67 (dilution 1:50) and PCNA (dilution 1:200) were carried out with the Vecta Elite kit (Vector Laboratories, Burlingame, CA, United States) based on the manufacturer’s manual. Sample analysis and image acquisition were achieved by Axio Scope A1 fluorescence microscope which was equipped with AxioCam MRC digital camera (Carl Zeiss, Oberkochen, Germany).
8
Immunohistochemical Staining of Tissue Samples
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The specimen sections were deparaffinized and rehydrated. Then, endogenous peroxidase was blocked using a hydrogen peroxide block (Abcam, UK). The specimens were washed, and antigen retrieval was performed by heating in a 10 mM citrate buffer. After protein block solution (Abcam, UK) was applied to reduce nonspecific background staining, the specimens were incubated with primary antibodies overnight at room temperature in a humidified chamber. The antigen-antibody complex was then detected using a Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC Kit (Abcam, UK), following the manufacturer's instructions. The sections were counterstained with hematoxylin solution (Mayer's modified, Abcam, UK) and dehydrated before mounting with mounting medium (Abcam, UK). As a negative control, the primary antibody was replaced with SignalStain antibody diluent (Cell Signaling Technology, USA). The stained specimens were examined under a Motic BA210 microscope.
9
Developmental Stages of UGP2 Expression in Fetal Brain
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For immunohistochemical analysis, we used two cases from the first trimester (GW6 and GW9), four cases from the second trimester (GW21, GW23, GW24 and GW26) and two cases from the third trimester (GW33 and GW36). Anatomical regions were determined according to the atlas of human brain development [11 –14 ]. We cut 4-µm sections from formalin-fixed, paraffin-embedded whole fetuses (GW6 and GW9) and brain tissue from cerebral, mesencephalic, cerebellar and brain stem regions (from GW21 to GW36). Slides were stained with mouse anti-UGP2 (C-6) in a 1:150 dilution (Santa Cruz) and visualized using Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit (Abcam). Mayer’s hematoxylin was used as a counterstain for immunohistochemistry followed by mounting and coverslipping (Bio-Optica) for slides. Prepared slides were analyzed and scanned under a VisionTek® Live Digital Microscope (Sakura).
10
Wnt Protein Expression in Mouse Lungs
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Mouse lung tissues were fixed in 4% paraformaldehyde for 48 h, dehydrated in ethanol and embedded in paraffin by using a TissueWaveTM Microwave Processor (Thermo Fisher Scientific). After dewaxing and rehydration, 5 μm-thick coronal sections were incubated in 0.01 M citrate buffer (pH 6.0) with 0.1% Tween-20 at 95–100°C for 10 min for antigen retrieval. For immunochemistry of Wnt2b and Wnt5b (n = 3 per group), the sections were incubated at 4°C overnight with primary antibody (Wnt2b at 1:200 or Wnt5b at 1:50). After being washed with PBST, the sections were stained using the mouse and rabbit-specific HRP/DAB (ABC) detection IHC kit (Abcam, ab64264) and analyzed using an Olympus BX60 compound microscope (Tokyo, Japan).
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