Serial sections of 4 µm were prepared from paraffin-embedded tissue previously fixed in neutrally buffered formalin and mounted on 3-aminopropyltriethoxysilane-coated slides. After overnight exposition at 50 °C, tissue samples were de-paraffinized and rehydrated in graded alcohol and xylol. Heat-mediated antigen retrieval was performed utilizing a water bath and 0.01 M sodium citrate for 30 min at 97 °С. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 for 5 min and subsequent washing with PBS. Sections were blocked with 1% BSA/PBS and incubated overnight at 4 °C with 0.1 µg/ml of monospecific anti-CEACAM1 (clone C5-1X/8; LeukoCom), anti-CEACAM5 (clone 3E10-3; LeukoCom) and anti-CEACAM6 (clone 1H7-4B; LeukoCom), or an isotype control. After washing, sections were probed with biotinylated secondary rabbit anti-mouse antibody (Dako) for 1 h at room temperature. Sections were washed and incubated with VECTASTAIN ABC reagent (Vector Laboratories) for 30 min according to the Manufacturers´ protocol. Staining was visualized by diaminobenzidine (DAB) substrate controlling the developing color intensity via light microscopy. DAB-negative structures were identified by additional counterstaining with hematoxylin. Stained sections were mounted and documented by microscopy.
Biotinylated rabbit anti mouse secondary antibody
Manufactured by Agilent Technologies
Sourced in Denmark
Biotinylated rabbit anti-mouse secondary antibody is a laboratory reagent used in various immunoassay techniques. It is designed to specifically bind to mouse primary antibodies, allowing for their detection and amplification in experimental procedures.
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Lab products found in correlation
6 protocols using biotinylated rabbit anti mouse secondary antibody
1
CEACAM Immunohistochemical Staining
2
Quantifying Epithelial Cell Proliferation using Ki-67
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Ki-67 is a cell cycle–associated antigen and regarded as a useful epithelial cell proliferation marker.39 (link) Distal colon segments were embedded in paraffin then sectioned at 4 µm. Sections were de-waxed in Histoclear and hydrogen peroxide (3%) was used for 15 minutes to quench the endogenous peroxidase activity. Antigen retrieval was achieved by cooking the sections in a pressure cooker at 120°C for 1 hour in 0.1 mol/L citrate buffer (pH 6.5). Sections were incubated with a primary monoclonal Ki-67 antibody (1:1000; Dako) at 4°C overnight. For detection of the primary antibody, biotinylated secondary rabbit-anti-mouse antibody (1:200; Dako) for 30 minutes and avidin/biotinylated peroxidase complex (Signet Laboratories USA-HRP kit) for 20 minutes were used. Slides were visualized by incubating with 3′-diaminobenzamine (DAB) substrate (Signet Laboratories USA-HRP kit) for 3 minutes, followed by counterstaining for 1 minute in hematoxylin and examining under a light microscope (Olympus, BH-2) at ×400 magnification. The percentage of proliferating cells was calculated in the same way as the apoptosis index described above.
3
Immunohistochemical Staining of Liver Tissue
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Staining for CD31+ and α-SMA was performed in cryosections from liver tissue (3μm and 7μm thickness, respectively) as described previously [51 (link)–53 (link)]. Briefly, after several steps, cryosections were incubated with a mouse anti-SMA antibody (clone 1A4; Sigma-Aldrich, Munich, Germany) or with antibody against CD31+ (ab24590, Abcam, Cambridge, UK). Thereafter, a biotinylated rabbit anti-mouse secondary antibody (Dako, Glostrup, Denmark) was used.
4
Immunohistochemistry of Hepatic Markers
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Stainings for CD31 and α-SMA were performed in cryosections from liver tissue (3 and 7 μm thick, respectively) as described previously41 (link)42 (link)43 (link). Briefly, after several steps cryosections were incubated with a mouse-anti-SMA antibody (clone 1A4; Sigma-Aldrich, Munich, Germany), or with antibody against CD31 (ab24590, Abcam, Cambridge, UK). Thereafter, a biotinylated rabbit-anti-mouse secondary antibody (Dako, Glostrup, Denmark) was used.
5
Histological Evaluation of Cartilage Markers
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The specimens (n = 4) were embedded into paraffin and sectioned at 5 µm. The sections were subjected to standard Safranin-O staining as previously described [28] (link). Immunostaining of Collagen I, II, X, and osteocalcin was performed as previously described [28] (link). Primary antibodies used were: Biotinlyated collagen I and II antibodies (1∶400, Chondrex, WA), mouse collagen X antibody (1∶500, Sigma, MO), mouse osteocalcin antibody (1∶500, Abcam, MA). For collagen X and osteocalcin detection, biotinylated rabbit anti-mouse secondary antibody (1∶1000, Dako, CA) was used. The sections with the same procedure but omitting the primary antibody were used as a negative control.
6
Double-stain Immunohistochemistry of Carnitine Acetyltransferase and Choline Transporters
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A mixture of mouse and rabbit primary antibodies (Table 4 ) was diluted in antibody diluent (Dako) and incubated with the sections overnight at 4 °C. After a 1-h treatment with alkaline-phosphatase-labeled swine anti-rabbit antibody (1:30; Dako), staining was visualized with Fast Blue chromogen. Subsequently, sections were incubated with biotinylated rabbit anti-mouse secondary antibody (1:150; Dako) and staining was visualized by using Vectastain ABC Elite Kit and DAB. Kaiser’s glycerol gelatine was used for cover-slipping. For control of staining, both antibodies were omitted or each primary antibody was omitted separately.
Antibodies used for double-stain immunohistochemistry with mouse and rabbit primary antibodies (CarAT carnitin acetyltransferase, CTL choline transporter-like)
| Primary rabbit antibody (blue) | Primary mouse antibody (brown) |
|---|---|
| Rabbit anti-human CarAT (1:100); LSBio (#LS-C167021/52635); preabsorption control with blocking peptide (LS-E8292/40 – LSBio) | Mouse anti-human CTL1 (1:100) |
| Rabbit anti-human CarAT (1:100); LSBio (#LS-C167021/52635); preabsorption control with blocking peptide (LS-E8292/40 – LSBio) | Mouse anti-human CTL2 (1:400) |
| Rabbit anti-human von Willebrand factor (1:10,000); Merck Millipore (#AB736) | Mouse anti-human CTL2 (1:400) |
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