To detect the number of macrophages and CD8+T cells, immunohistochemistry was performed. Paraffin‐embedded, 3‐µm‐thick skin tissue sections were stained for CD68 (1:100, clone PG‐M1, Dako, M087629‐2) and CD8alpha (1:150, clone 144B, Abcam, ab17147) using the Mouse specific HRP/DAB Detection IHC Kit (Ab64259, Abcam), according to the manufacturer's protocol. In brief, after deparaffinization, tissue sections were blocked for endogenous peroxidases using hydrogen peroxide for 10 minutes. Antigen retrieval was performed by HIER (microwave in TE buffer pH 8.0), and blockage of unspecific binding was done by incubating sections with protein block for 10 minutes. Between each step, buffer washes using TBST (5 mmol/L Tris‐HCl (pH 7.6), 150 mmol/L NaCl, and 0.1% (v/v) Tween‐20) were interposed. Incubation with primary antibody was done overnight (12‐14 hours) at 4°C. After washing, the sections were incubated with secondary antibody for 10 minutes. The reactions were visualized with DAB and counterstained with Mayer's Htx (Dako).
Mouse specific hrp dab detection ihc kit
Manufactured by Abcam
The Mouse Specific HRP/DAB Detection IHC Kit is a laboratory tool designed for immunohistochemical (IHC) staining of mouse-derived tissue samples. The kit provides the necessary components, including the HRP (horseradish peroxidase) enzyme and the DAB (3,3'-diaminobenzidine) chromogen, to enable the specific detection of target antigens in mouse tissue sections.
Automatically generated - may contain errors
Lab products found in correlation
Ab49777, by Abcam (2 mentions)
Clone 144b, by Abcam (1 mentions)
Clone pg m1, by Agilent Technologies (1 mentions)
Ab79056, by Abcam (1 mentions)
Ab6671, by Abcam (1 mentions)
Ab183685, by Abcam (1 mentions)
Sc 20047, by Santa Cruz Biotechnology (1 mentions)
A microscope, by Leica Microsystems (1 mentions)
Ab7817, by Abcam (1 mentions)
5 protocols using mouse specific hrp dab detection ihc kit
1
Quantifying Immune Cells in Skin Tissue
2
Immunohistochemical Profiling of Mouse Lung
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For immunohistochemical staining, paraffin-embedded sections (4 μm) of mouse lung tissue were used. Heat-mediated antigen retrieval in citrate buffer (pH = 6.0) was performed. Deparaffinized tissue-sections were incubated with primary mouse anti-CD3 (sc-20047, Santa Cruz Biotechnology), anti-CD4 (ab183685, Abcam), anti-CD68 (ab49777, Abcam), anti-TNF-α antibody (ab6671, Abcam), and anti-IL-17 (ab79056, Abcam). Staining was visualized by using Mouse Specific HRP/DAB Detection IHC Kit (ab64259, Abcam) for CD3 and CD68, and rabbit specific HRP/AEC detection IHC Kit (ab94361, Abcam) for CD4, TNF-α, and IL-17. Sections were counterstained with Mayer's hematoxylin. Sections were photomicrographed with a digital camera mounted on light microscope (Olympus BX51, Japan), digitized, and analyzed.
3
Histological Analysis of Pancreatic Islets
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The isolated pancreas was fixed in 10% neutral buffered formalin, dehydrated using a series of graded alcohols, and embedded in paraffin. Sections (2 μm in thickness) were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). Four H&E-stained sections per mouse were used for measuring islet area using the ImageJ software (National Institutes of Health, NIH). For microscopy imaging of insulin, glucagon, and CD68, deparaffinized pancreas sections were incubated with primary antibodies (Abcam, Cambridge, UK) overnight at 4 °C. Staining was visualized using the mouse-specific HRP/DAB detection IHC kit (Abcam). Images of islets were obtaineded using a microscope (Leica Microsystems, Wetzlar, Germany). The percentage of target signal-positive area was calculated by dividing the area of target signal by the total islet area for at least 10 islets per mouse.
4
Islet Morphometry and Immunohistochemistry
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The isolated pancreas was fixed in 10% neutral-buffered formalin, dehydrated using a graded series of alcohol, and embedded in paraffin. Sections (2 μm in thickness) were deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). Four H&E-stained sections per mouse were used for measuring islet area using ImageJ software (Version 1.8.0, National Institutes of Health, NIH, Bethesda, MA, USA). For microscopy imaging of insulin and glucagon, deparaffinized pancreas sections were incubated with primary antibodies (Abcam, Cambridge, UK) overnight at 4 °C. Staining was visualized using mouse-specific HRP/DAB detection IHC kit (Abcam). Images of islets were acquired using a microscope (Leica Microsystems, Wetzlar, Germany). The percentage of target signal-positive area was calculated by dividing the area of target signal by the total islet area for at least 10 islets per mouse.
5
Immunohistochemical Analysis of Liver Tissue
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For immunohistochemical staining we used paraffin embedded sections (5μm) of mouse liver tissue. We performed heat mediated antigen retrieval in Citrate Buffer (pH = 6.0). Deparaffinized tissue-sections were incubated with primary mouse anti-α-SMA antibody (ab7817, Abcam, Cambridge, UK) and primary mouse anti-CD68 antibody (ab49777, Abcam). Staining was visualized by using Mouse Specific HRP/DAB Detection IHC Kit (ab64259, Abcam) and sections were counterstained with Mayer's hematoxylin. Sections were photomicrographed with a digital camera mounted on light microscope (Olympus BX51, Japan), digitized and analyzed. Analysis was performed on 10 fields of a section at 40X magnification. Results are presented as mean count of positive cells per field.
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