The procedure was carried out as previously published [13 (link)]. In brief, tissue sections were dewaxed for 2 × 15 min in Histoclear (National Diagnostics), rehydrated and endogenous peroxidase activity blocked with 1% H2O2 (Sigma, Darmstadt, Germany) in methanol (VWR UK) for 10 min. Antigen retrieval (in 0.01 M, pH 6 citric acid buffer for 2 min), preceded blocking of background staining with 10% BSA (Sigma, Darmstadt, Germany) for 1 h. Sections were incubated for 1 h with primary antibody (NESOpAb), specific for nNav1.5 [19 (link)], at a dilution of 1:200 from a stock concentration of 0.7 mg/mL. Following washing, biotinylated swine anti-rabbit secondary antibody (DAKO) at a 1:125 dilution was applied for 1 h. All antibody incubations were performed at room temperature. Following completion of the ABC kit protocol DAB staining (Vector Laboratories Inc., Burlingame, CA, USA) and DAB staining (Vector Laboratories Inc.), sections were haematoxylin stained and mounted with DPX. In negative controls, the primary antibody was omitted which eliminated the staining (not shown). Nuclei were counterstained in blue by haematoxylin. Immunohistochemistry for the other biomarkers was performed as previously reported [30 (link),31 (link),32 (link)].
Dab staining
Manufactured by Vector Laboratories
Sourced in United States
DAB staining is a laboratory technique used to detect the presence of specific proteins or other molecules within tissue samples. It involves the application of a chemical substrate that produces a brown stain when it interacts with the target analyte, allowing for visualization and identification of the desired target.
Automatically generated - may contain errors
Lab products found in correlation
Rat anti mouse f4 80, by Thermo Fisher Scientific (2 mentions)
Sirius red, by Abcam (2 mentions)
Rabbit anti mouse α sma, by Abcam (2 mentions)
Anti αsma ab, by Abcam (2 mentions)
Rabbit anti mouse desmin, by Thermo Fisher Scientific (2 mentions)
Rabbit anti mouse 4 hne, by Alpha Diagnostic (2 mentions)
Vectastain abc kit, by Vector Laboratories (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Methanol, by Avantor (1 mentions)
Histoclear, by National Diagnostics (1 mentions)
Osteosoft, by Merck Group (1 mentions)
Axiovert s100 tv, by Zeiss (1 mentions)
Anti nestin, by Merck Group (1 mentions)
Axiovert 100 inverted microscope, by Zeiss (1 mentions)
Anti gfap, by Novus Biologicals (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Secondary anti rabbit igg, by Vector Laboratories (1 mentions)
21 protocols using dab staining
1
Immunohistochemical Detection of Sodium Channel
2
Immunohistochemical Analysis of YAP1 in Transgenic Pig Samples
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Immunohistochemistry was performed as described previously [44 (link)]. OS samples (n = 6) from flTP53R167H homozygous pigs were fixed in 4% formalin and decalcified in Osteosoft® (Merck, Darmstadt, Germany). Four-micrometer sections were air-dried for 10 min at 60 °C on a glass slide. Antigen demasking was performed using the heat retrieval procedure (20 min, citrate buffer pH 6, pressure cooker in microwave medium intensity). Sections were stained with biotinylated rabbit anti-YAP1 antibody (diluted 1:200; ARP50530_P050, Aviva System Biology Cooperation, San Diego, CA, USA) and binding visualized with the avidin-peroxidase solution (ABC kit, Vector, Darmstadt, Germany) followed by DAB staining (Vector). Sections were lightly counterstained with haematoxylin (Merc, Darmstadt, Germany). Pig duodenum sections were used as a positive control. No incubation with primary antibody was used as a negative control.
3
Immunohistochemical Analysis of REST Protein
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Immunohistochemical analyses of normal and tumor samples were performed by Dr. Veena Rajaram following Institutional Review Board (IRB) approval. Paraffin-embedded brain sections were immunohistochemically stained by hematoxylin eosin (H&E), and REST protein expression was analyzed using anti-REST antibody (Cat# IHC-00141; Bethyl, Montgomery, TX; 1:100 dilution) followed by DAB staining (Vector laboratories, Burlingame, CA). Slides were evaluated for REST levels by neuropathologist utilizing a 5-point grading scale as described in Figure 1A .
4
Liver Fibrosis Immunohistochemistry Analysis
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Formalin-fixed frozen livers were stained with Sirius Red and anti-α-SMA Ab (Abcam). Immunohistochemistry was performed using DAB staining (Vector), and counterstaining with Hematoxilin. The following antibodies were used for immunostaining: rabbit anti-mouse α-SMA (Abcam, Cambridge, MA), rabbit anti-mouse Desmin (Thermo Fisher Scientific, Fremont, CA), rat anti-mouse F4/80 (eBioscience, San Diego, CA), or rabbit anti-mouse 4-HNE (Alpha Diagnostic Intl Inc., San Antonio, TX) antibodies, following incubation with Alexa Fluor ® secondary antibody. The images were taken using Nikon microscope, and analyzed by Image J.
5
Immunohistochemical Analysis of Brain Tissue
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For immunohistochemical analysis, tissue sections were de-paraffinized, rehydrated and subjected to high temperature antigen retrieval in 10 mM citrate buffer (pH 6.0). The following primary antibodies were used: rabbit monoclonal anti-Ki67 (clone SP6, Lab Vision, 1:100) rabbit polyclonal anti-GFAP (Novus Biologicals, 1:100), anti-nestin (Millipore, 1:100). These primary antibodies were applied for 2 h in blocking buffer (2.5% BSA, 5% goat serum, 0.3% Triton X-100 in PBS), followed by species-appropriate secondary Alexa Fluor 488 dye conjugated antibodies (Amersham) or Vectastain ABC kit and DAB reagents (Vector Laboratories). Fluorescence antibody-labeled slides were mounted in DAKO fluorescent mounting medium containing 1 μg ml−1 Hoechst counterstain. HRP-conjugated secondary antibodies were visualized by DAB staining (Vector Laboratories). Apoptotic cells were detected with ApopTag Detection kit (Chemicon International). Images were obtained with an Axiovert S100 TV inverted fluorescence microscope (Zeiss) and Open Lab 3.5.1 software, or with an Axiovert 100 inverted microscope (Zeiss) equipped with a Hamamatsu Orca digital camera.
6
Immunohistochemical Analysis of CD68+ Cells
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Formalin fixed knee joints were decalcified in 8% formic acid (Sigma-Aldrich, Milan, Italy). All tissues were dehydrated, embedded in paraffin, and sectioned into 7 μm sections. For the CD68 staining, knee joint sections were rehydrated and digested for 1 h at room temperature with 0.05% hyaluronidase (Sigma-Aldrich, Milan, Italy). Bone and tissue sections were incubated for 1 h with blocking solution (1× PBS, 0.5% Tween-20, 0.1% bovine serum albumin and 10% fetal bovine serum, GIBCO BRL–Invitrogen, Gaithersburg, MD, USA) and incubated overnight with a polyclonal anti-CD68 antibody (1:300 diluted, Serotec, Oxford, UK). After washing, sections were incubated for 1 h with biotinilated secondary anti-rabbit IgG (Vector laboratory, CA, USA). The reaction was developed using the Vectastained Elite ABC-Peroxidase Kit (Vector laboratory, CA, USA), followed by a 30 min DAB staining (Vector laboratory, CA, USA). Finally, sections were counterstained with hematoxylin (Sigma-Aldrich, Milan, Italy) and mounted with Eukitt (Kaltek, Padova, Italy).
7
Histomorphometric Analysis of Adipose Tissue
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Fat pads fixed in 10% formalin were embedded in paraffin and sectioned. Specimens were then stained by hematoxylin and eosin, and cell area was measured using National Institutes of Health (NIH) ImageJ software (http://imagej.nih.gov ). Sections of adipose tissue samples were immunohistochemically stained by using primary antibodies to UCP1 (Abcam) and Vectastain ABC kit (Vector Laboratories) according to the manufacturers’ instructions. Peroxidase activity was visualized with DAB staining (Vector Laboratories), and sections were counterstained with hematoxylin.
8
Histological Evaluation of Mouse Kidney Fibrosis
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Paraffin-embedded mouse kidney sections (3-µm thickness) were stained with PAS (catalog no. G1280; Solarbio, Beijing, China), Masson (HT15-1KT; Sigma–Aldrich, St Louis, MI, USA), and Sirius red (catalog no. G1472-2; Solarbio, Beijing, China). The antibodies for immunohistochemistry were: anti-α-SMA (catalog no ab32575, Abcam, Cambridge, United Kingdom), anti-fibronectin (catalog no. 610154; Transduction Laboratories, Lexington, KY, USA), anti-type I collagen (catalog no. AB765P; Millipore Corp., Billerica, MA, USA) and anti-CCR2 (catalog no. ab176390; Abcam, Cambridge, United Kingdom) and anti-galectin-3 (catalog no. 3027070; Millipore Corp., Billerica, MA, USA). After incubation with the primary antibodies at 4 °C overnight, the slides were stained with the secondary antibody for 1 h at room temperature. The sections were incubated with the ABC reagents for 1 h at room temperature before DAB staining (Vector Laboratories, Burlingame, CA, USA). Images were captured using a light microscope (Olympus, Tokyo, Japan).
9
Quantifying Amyloid Plaques and Microglia
10
Immunohistochemical Analysis of Multiple Sclerosis Lesions
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Snap frozen tissue was sectioned (10-μm), fixed (4% PFA), blocked in PBS with 10% normal horse serum (NHS) and stained for myelin oligodendrocyte glycoprotein (MOG) (R. Reynolds, Imperial College, UK) and HLA-DR (Dako UK Ltd) followed by biotinylated secondary antibody (Jackson Immunoresearch Laboratories, Cambridgeshire, UK), avidin/biotin staining (Vector Laboratories, Burlingame, CA) and DAB staining (Vector Laboratories, Burlingame, CA). All sections were also stained with haematoxylin-eosin (H&E) (R. Reynolds, Imperial College, UK). The five pathological areas (NAWM, active, chronic active, inactive and repairing/early remyelinating lesions) were characterized based on MOG+ staining showing myelin integrity and HLA-DR+ staining showing the inflammatory state, and each lesion type was defined as described (Reynolds et al., 2011). All antibodies and concentration are listed in Additional file 1 . Quantification of CD20+ cells were performed in four active and three remyelinating lesions from seven different patients. Ten pictures depending on size per lesion type were taken at an objective lens magnification of 20x. The CD20+ cells were manually counted, and the average number of cells per lesion from each patient was compared by using Mann-Whitney Test performed in Graphpad Prism.
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