Jejunum and ileum sections were fixed in 4% paraformaldehyde and embedded in paraffin as described [22 (link)]. Mast cells in the jejunum were visualized using the Naphthol AS-D chloroacetate esterase staining kit (Sigma-Aldrich). Eosinophils in the jejunum and Foxp3+T cells in the ileum were identified by immunohistochemistry (IHC) by incubating tissue sections with anti-mouse major basic protein monoclonal antibody (1:1500; provided by Jamie J. Lee, Mayo Clinic, Scottsdale, AZ, USA) and anti-mouse/rat Foxp3 monoclonal antibody (clone FJK-16s; 1:100; eBioscience), respectively. The sections were then developed with HRP conjugated goat anti-rat IgG antibody (Millipore, Billerica, MA, USA), DAB color-developing solution (Dako, Glostrup Municipality, Denmark), and counter-stained with hematoxylin gill number 3 (Sigma-Aldrich). For all analyses, two tissue sections were stained. Quantification of mast cells and eosinophils was performed by analyzing three random sections per stained sample on the ImageJ software and expressed as number of cells per mm2 of mucosa.
Hrp conjugated goat anti rat igg antibody
Manufactured by Merck Group
Sourced in United States, Australia, Germany
The HRP-conjugated goat anti-rat IgG antibody is a laboratory reagent that can be used to detect and quantify rat immunoglobulin G (IgG) in various immunoassays and research applications. The antibody is conjugated with horseradish peroxidase (HRP), an enzyme that can catalyze a colorimetric or chemiluminescent reaction, allowing for the visualization and quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
F4 80, by Abcam (3 mentions)
Naphthol as d chloroacetate esterase staining kit, by Merck Group (1 mentions)
Dab color developing solution, by Agilent Technologies (1 mentions)
Hrp conjugated goat anti rabbit igg antibody, by Merck Group (1 mentions)
Biozero software, by Keyence (1 mentions)
Dab substrate, by Agilent Technologies (1 mentions)
Fitc conjugated goat anti rabbit igg, by Abcam (1 mentions)
5 protocols using hrp conjugated goat anti rat igg antibody
1
Histological Analysis of Intestinal Immune Cells
2
Analyzing Macrophage and Nox4 Levels in Kidneys
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Immunoperoxidase staining was performed as previously described [17] (link). In brief, macrophage infiltration was analyzed using a monoclonal antibody for murine monocyte/macrophage (F4/80, Abcam), followed by HRP-conjugated goat anti-rat IgG antibody (Millipore). The number of F4/80-positive cells was calculated in 10 glomeruli and intestitia per animal, and the mean number of F4/80 positive cells per glomerulus and interstitial tissue (number per mm2) was used for the estimation.
NADPH oxidase 4 (Nox4) immunoperoxidase staining was performed as described previously [19] (link). Briefly, renal tissues were stained with Nox4 rabbit antibody (Novus Biologicals, Littleton, CO, USA) for 12 h at 4°C followed by HRP-conjugated goat anti-rabbit IgG antibody (Millipore). The proportion of the area stained with Nox4 antibody of the total area was calculated using BIOZERO software (Keyence). To quantify the proportional area of staining, 10 views of the renal cortex were randomly selected in each slide.
NADPH oxidase 4 (Nox4) immunoperoxidase staining was performed as described previously [19] (link). Briefly, renal tissues were stained with Nox4 rabbit antibody (Novus Biologicals, Littleton, CO, USA) for 12 h at 4°C followed by HRP-conjugated goat anti-rabbit IgG antibody (Millipore). The proportion of the area stained with Nox4 antibody of the total area was calculated using BIOZERO software (Keyence). To quantify the proportional area of staining, 10 views of the renal cortex were randomly selected in each slide.
3
Immunoperoxidase Staining for Macrophage Quantification
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Immunoperoxidase staining was performed as previously described.50 (link) In brief, macrophage infiltration was analyzed using a monoclonal murine monocyte/macrophage antibody (F4/80; Abcam), followed by an HRP-conjugated goat anti-rat IgG antibody (Merck KGaA). The number of F4/80-stained cells was evaluated similarly to the neutrophil counts.
4
Dual Immunostaining for Macrophages and Neutrophils
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Renal sections were incubated with rat anti-mouse F4/80 antibody (1 h, 20°C), washed in TBST (1 min) then treated with an HRP-conjugated goat anti-rat IgG antibody (Merck Millipore, Kilsyth, Australia) for 30 min. Slides were then washed with TBST (1 min) and the developed (20°C, 6 min) with DAB substrate (Dako, North Sydney, Australia).
Sections were then stained for MPO, a heme enzyme present in neutrophils and macrophages. Mounted renal specimens were incubated with rabbit polyclonal anti-MPO antibody (1 h), washed in TBST and incubated with FITC-conjugated goat anti-rabbit IgG (Abcam Cat#ab97050), in a humid environment (30 min, 20°C). Slides were then washed with TBST (4x 5 min) and dipped in Sudan Black B solution (1% v/v, 3 min) to reduce background autofluorescence.
Slides were left in TBST wash solution overnight before counterstaining with DAPI. Images were digitally processed with Photoshop CS2 software (Adobe Systems, Sydney, Australia). DAB-stained images were processed to mimic red immunofluorescence, by digitally subtracting the blue color channel from the red color channel using the Apply Image tool. The processed color channel was then superimposed over DAPI and FITC channels to form the final image as described previously (33 (link)).
Sections were then stained for MPO, a heme enzyme present in neutrophils and macrophages. Mounted renal specimens were incubated with rabbit polyclonal anti-MPO antibody (1 h), washed in TBST and incubated with FITC-conjugated goat anti-rabbit IgG (Abcam Cat#ab97050), in a humid environment (30 min, 20°C). Slides were then washed with TBST (4x 5 min) and dipped in Sudan Black B solution (1% v/v, 3 min) to reduce background autofluorescence.
Slides were left in TBST wash solution overnight before counterstaining with DAPI. Images were digitally processed with Photoshop CS2 software (Adobe Systems, Sydney, Australia). DAB-stained images were processed to mimic red immunofluorescence, by digitally subtracting the blue color channel from the red color channel using the Apply Image tool. The processed color channel was then superimposed over DAPI and FITC channels to form the final image as described previously (33 (link)).
5
Macrophage Infiltration Analysis in Kidney Sections
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