To immunostain CD31, tumor sections were fixed at 4°C for 10 min in 4% paraformaldehyde, immersed in 1% H2O2 at room temperature for 30 min to quench endogenous peroxidases, and blocked at room temperature for 30 min with Blocking One (Nacalai tesque; Kyoto, Japan). Sections were then probed at 4°C overnight with 1:500 anti-CD31 (PECAM-1; BD Biosciences; Franklin Lakes, NJ), washed in Tris-buffered saline, and labeled at room temperature for 45 min with 1:400 biotinylated rabbit anti-rat (DAKO), and then at room temperature for 45 min with LSAB (DAKO). Finally, sections were stained with 3,3-diaminobenzidine (DAKO) and hematoxylin (Wako).
Biotinylated rabbit anti rat
Manufactured by Agilent Technologies
Sourced in Denmark
Biotinylated rabbit anti-rat is a reagent used in various immunoassay and immunohistochemistry applications. It consists of rabbit-derived antibodies that have been labeled with biotin, a small molecule that can be detected and used to signal the presence of the antibody.
Automatically generated - may contain errors
Lab products found in correlation
Blocking one, by Nacalai Tesque (2 mentions)
Hematoxylin, by Fujifilm (1 mentions)
Pecam 1, by BD (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Rat monoclonal anti brdu, by Abcam (1 mentions)
Mouse monoclonal anti tyrosine hydroxylase, by Merck Group (1 mentions)
Anti calretinin, by Merck Group (1 mentions)
Anti neun, by Merck Group (1 mentions)
Proteinase k, by Roche (1 mentions)
Anti cd31 pecam 1, by BD (1 mentions)
Anti mouse cd31 pecam 1, by BD (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
Biotinylated horse anti mouse, by Vector Laboratories (1 mentions)
Alexa 488 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Streptavidin conjugated alexa 568, by Thermo Fisher Scientific (1 mentions)
Anti ki67, by BD (1 mentions)
Sheep anti mouse igd, by Abcam (1 mentions)
3 3 diaminobenzidine tablets, by Merck Group (1 mentions)
Rat anti mouse cd3, by Bio-Rad (1 mentions)
7 protocols using biotinylated rabbit anti rat
1
Immunohistochemical Detection of CD31
2
Quantifying Olfactory Neuron Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were perfused transcardially with 4% paraformaldehyde. For immunohistochemistry, the following primary antibodies were applied to 50 μm free-floating coronal OB sections: mouse monoclonal anti-tyrosine hydroxylase (TH) (1:500), anti-calretinin (CR) (1:1000) and anti-NeuN (1:200) from Millipore, mouse monoclonal anti-calbindin (CB) (1:500) from Sigma, rat monoclonal anti-BrdU (1:200), rabbit monoclonal pSer129 α-SYN (1:500) from Abcam, and rat monoclonal antibody 15G7 against human α-SYN (116–131) peptide MPVDPDNEAYEMPSEE (1:4000) from Connex31 . As secondary antibodies, we used: goat anti-mouse Alexa 488/647 (1:500), rabbit anti-rat Alexa 488/647 (1:500) from Invitrogen and biotinylated rabbit anti-rat (1:300) from Dako. Enzymatic digestion using Proteinase K (PK; Roche) was employed for 8–10 min. Peroxidase detection was applied for 8–10 min (DAB detection kit, DCS Diagnostics). Sections were imaged at 0.75 μm pixels resolution in xy dimension and 1 μm in z dimension on a confocal microscope (Zeiss LSM 510) equipped with a 10×, 25× or 40× oil objective (Zeiss). Counting of somata and nuclei was performed manually from confocal image stacks, using Zeiss AIM software. For consistent analysis, every tenth OB slice was used and imaged at predefined positions within the glomerular layer.
3
Quantifying Tumor Angiogenesis via CD31
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor sections were fixed in 4% paraformaldehyde at 4°C for 10 minutes, immersed in 1% H2O2 at room temperature for 30 minutes to quench endogenous peroxidases, and blocked at room temperature for 30 minutes with Blocking One (Nacalai Tesque). Sections were then probed at 4°C overnight with 1:500 anti‐CD31 (PECAM‐1; BD Biosciences), washed in TBS, labeled at room temperature for 45 minutes with 1:400 biotinylated rabbit anti‐rat (Dako), and then labeled at room temperature for 45 minutes with streptavidin‐biotin (Dako). Sections were stained with 3,3‐diaminobenzidine and hematoxylin. Harvested s.c. tumors were immunostained with anti‐mouse CD31 (PECAM‐1; BD Biosciences). The number of stained microvessels was counted at 400× in 4 random fields for each tumor.
4
Multimarker Immunohistochemistry of Pancreatic Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunohistochemical detection of VDR, insulin, glucagon, somatostatin, pancreatic polypeptide, TUNEL, and Ki67, pancreas were fixed for 12–24 h in formalin, embedded in paraffin, and sectioned. Sections were then incubated overnight at 4°C with the following antibodies: rat anti-mouse VDR (clone 9A7; Merck KGaA, Darmstadt, Germany), guinea pig anti-porcine insulin (Sigma Chemical, St Louis, MO), rabbit anti-human glucagon (Signet Laboratories, Dedham, MA), rabbit anti-somatostatin (Serotec, Oxford, U.K.), rabbit anti-human pancreatic polypeptide (ICN Biomedicals), and anti-Ki67 (BD Pharmingen). As secondary antibodies, peroxidase-conjugated rabbit anti-guinea pig IgG (Dako, Glostrup, Denmark), biotinylated goat anti-rabbit (Pierce, Rockford, IL), tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-guinea pig (Molecular probes, Leiden, the Netherlands), biotinyilated goat anti-rabbit (Molecular Probes), biotinylated rabbit anti-rat (Dako), and biotinylated horse anti-mouse (Vector Laboratories, Burlingame, CA) antibodies were used. Streptavidin-conjugated Alexa 488 (Molecular Probes) or streptavidin-conjugated Alexa 568 (Molecular Probes) were used as fluorochromes. Images were obtained with a Nikon Eclipse 90i microscope (Nikon, Tokyo, Japan).
5
Spleen Tissue Immunohistochemistry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spleens were processed and stained as described previously (32 (link)). In short, spleens were frozen in liquid nitrogen and 5.5-μm-thick sections were cut and allowed to dry before fixation in acetone for 20 min at 4°C. In all cases, antibodies were added for 45 min of incubation at room temperature. Sheep anti-mouse IgD (Abcam) was detected with peroxidase-labeled donkey anti-sheep antibody (Jackson ImmunoResearch, Inc.) and developed with 3,3′-diaminobenzidine tablets (Sigma). CD3 and peanut agglutinin (PNA) were detected with rat anti-mouse CD3 (AbD Serotec), biotinylated rabbit anti-rat (Dako), and biotinylated rabbit anti-PNA (Vector Laboratories) antibodies. Biotinylated Ab binding signal was developed by using a streptavidin-biotinylated AP complex (Vectastain; Vector Laboratories) with Naphthol AS-MX phosphate, levamisole, and Fast Blue Salt.
6
Immunohistochemical Analysis of Mouse Eye Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice were killed with an overdose of CO2, the eyes were immediately immersed in OCT medium, snap frozen in cooled isopentane on dry ice, and stored at −80°C until used. Primary antibody was placed for 1 hour at room temperature on 6-μm thick eye sections, followed by secondary biotinylated rabbit anti-rat (DakoCytomation, Glostrup, Denmark) or biotinylated mouse anti-hamster Ig cocktail (BD Pharmingen, Oxford, UK) for 30 minutes at room temperature followed by Vectastain ABC-AP kit (Vector Laboratories, Peterborough, UK) for 30 minutes. For confocal imaging secondary fluorescent-labelled antibodies Alexa Fluor 555 goat anti-rat IgG (Invitrogen, Waltham, MA, USA) and Alexa Fluor 488 goat anti-rat IgG (Invitrogen) were used together with nuclear staining 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Peterborough, UK). The following primary monoclonal antibodies (mAb) were used in concentrations as suggested by the manufacturer: rat anti-mouse CD11b (clone M1/70); rat anti-mouse CD4 (clone H129.19), rat anti-mouse CD8α (clone 53-6.7), rat anti-mouse Gr-1 (clone RB6-8C5), hamster anti-mouse CD11c (clone HL3; all from BD Pharmingen) and rat anti–mouse F4/80 (clone C1:A3-1; AbD Serotec, Kidlington, UK).
7
Immunohistochemical Analysis of Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin sections (10 μm) were dewaxed and stained histochemically with hematoxylin and eosin (HE). For immunohistochemistry, the paraffin sections were dewaxed and subjected to a heat-induced epitope retrieval step except for sections for prior incubation with anti-B220 (clone RA3-6B2, BD Bioscience, 1:400). Primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, USA, 1:400) and MPO7 (polyclonal rabbit, Dako, code A0398, 1:1000) were used. This was followed by incubation with biotinylated secondary antibodies (Dianova). For detection, alkaline phosphatase-labelled streptavidin and chromogen RED (both Dako) were employed. For detection of macrophages, sections were subjected to protein-induced epitope retrieval employing protease (Sigma) prior to incubation with anti-F4/80 (clone BM8, eBioscience, 1:800). This was followed by incubation with biotinylated rabbit anti-rat (Dako) secondary antibody. Biotin was detected using alkaline phosphatase-labelled streptavidin (Dako). For visualization of alkaline phosphatase, chromogen RED (Dako) was used. Nuclei were counterstained with hematoxylin (Merck). Negative controls were performed by omitting the primary antibody.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!