Mice were deeply anaesthetised by i.p. injection of pentobarbital and perfused transcardially with phosphate buffered saline (PBS), followed by ice-cold 4% (w/v) paraformaldehyde/PBS. The L4 segments of the spinal cord, or the L4 DRG were removed, postfixed in the same fixative for 3 h at 4 °C, and placed in 30% (w/v) sucrose solution for 24 h at 4 °C. Transverse L4 spinal cord sections (30 μm) and L4 DRG sections (15 μm) were incubated in blocking solution [3% (v/v) normal goat serum] for 2 h at room temperature and then incubated for 48 h at 4 °C with primary antibodies: rabbit polyclonal anti-Iba1 (1:5000, Wako), and rat monoclonal anti-CD11b (1:1000, Serotec), rat monoclonal anti-CD31 (1:200, BD Pharmingen) and hamster monoclonal anti-CD3 (1:100, eBioscience). Following incubation, tissue sections were washed and incubated for 3 h at room temperature in secondary antibody solution (Alexa Fluor 546 and Alexa Fluor 405, 1:1000, Molecular Probes, OR, USA). The tissue sections were washed, slide-mounted and subsequently coverslipped with Vectashield hardmount (Vector Laboratories). Three to five sections from the L4 spinal cord and DRG of each mouse were randomly selected and analysed using an LSM700 Imaging System (Carl Zeiss).
Rat monoclonal anti cd31
Manufactured by BD
Sourced in United States
Rat monoclonal anti-CD31 is a laboratory research reagent used to detect the presence of the CD31 (PECAM-1) protein. CD31 is a cell surface glycoprotein expressed on endothelial cells and some leukocytes. This antibody can be used to identify and study cells expressing CD31.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 405, by Thermo Fisher Scientific (1 mentions)
Rat monoclonal anti cd11b, by Bio-Rad (1 mentions)
Vectashield hardmount, by Vector Laboratories (1 mentions)
Lsm700 imaging system, by Zeiss (1 mentions)
Rabbit polyclonal anti iba1, by Fujifilm (1 mentions)
Alexa fluor 647 conjugated goat anti rat igg h l, by Thermo Fisher Scientific (1 mentions)
Alexa 594 donkey anti rat, by Thermo Fisher Scientific (1 mentions)
Donkey anti rat alexa 488, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse alexa 594, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Fluorescence microscopy, by Olympus (1 mentions)
Ziess axiocam digital camera, by Zeiss (1 mentions)
Tissue tek, by Thermo Fisher Scientific (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Cm3050s cryostat, by Leica (1 mentions)
Ab89901, by Abcam (1 mentions)
Ab15580, by Abcam (1 mentions)
Alexa fluor antibodies, by Thermo Fisher Scientific (1 mentions)
5 protocols using rat monoclonal anti cd31
1
Immunohistochemical Analysis of Spinal Cord and DRG
2
Whole-Mount Retina and Choroid Immunostaining
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Enucleated eyes from 8-week-old C57BL/6J mice were fixed for 30 minutes in 4% paraformaldehyde (PFA) in PBS and then dissected. Retinal cups were removed before immunostaining the whole-mount retina. To whole-mount immunostain the choroid, the RPE/choroid/sclera complex was additionally fixed with 4% PFA for 30 minutes, and the RPE was removed by gentle brushing, resulting in a choroid/sclera complex. The retinal cups and choroid/sclera complexes were stained with primary antibodies, including rat monoclonal anti-CD31 (1:200, BD, cat. #553370) and PE anti-mouse CD157 antibody (clone BP-3, BioLegend, cat. #140204). The secondary antibody used was Alexa Fluor 647-conjugated goat anti-Rat IgG (H+L) (1:500, Thermo Fisher, cat. #A21247).
3
Immunostaining of Mouse Tissue Sections
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Mice were anesthetized with 30% urethane. After cardiac perfusion with PBS, tissues were removed and fixed in 4% paraformaldehyde (PFA) overnight. After dehydration in 30% sucrose, tissues were embedded in OCT and frozen, and tissue blocks were stored at -80°C until sectioning. 10 μm serial sections were obtained for immunostaining. After blocking tissue sections with bovine serum albumin (BSA), sections were incubated with biotin-plCSA-BP (50 ng/mL), biotin-SCR (50 ng/mL) or primary antibodies overnight at 4°C as follows: rat monoclonal anti-CD31 (1:200; BD Pharmingen, CA, USA); rat monoclonal anti-cytokeratin 8 (CK8; 1:100; National Institutes of Health Developmental Studies Hybridoma Bank, Iowa, USA); or mouse monoclonal anti-C4S (2B6, 1:20; Amsbio, UK). Tissue sections were then washed 3 times (TBS, 5 min) and incubated at room temperature with Alexa 488-donkey anti-rat (1:200; Thermo Fisher Scientific, MA, USA), Alexa 594-donkey anti-rat (1:200; Thermo Fisher Scientific, MA, USA), or Alexa 594-goat anti-mouse (1:200, Thermo Fisher Scientific, MA, USA) secondary antibody or FITC-avidin (1:64; Boster, Wuhan, China) separately for 30 min. Stained sections were mounted using mounting medium containing DAPI (Vector Laboratories, CA, USA) and examined using fluorescence microscopy (Olympus, Japan) and confocal microscopy (Leica, TCS SP5, Germany).
4
Quantifying Skeletal Muscle Capillary Density
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Following published procedures from our lab28 , muscle complexes were divided at the midline along the axial plane and embedded in Tissue-Tek (Fisher Scientific). Three transverse sections per sample were cut for each histological assessment using a CM3050S cryostat (Lecia, Wezlar, Germany). Sections were placed on frozen microscope slides and stored at − 80 °C before staining with rat monoclonal anti-CD31 (BD, San Diego, CA), a marker for endothelial cells. Samples were co-stained with mouse monoclonal anti-dystrophin (MANDRA1) (Sigma-Aldrich, St. Louis, MO) to outline myofibers and measure skeletal muscle area, necessary for calculation of capillary density. For details on immunohistochemistry, see Supplementary Methods. Adobe Photoshop CS5 was used to analyze 5 digital images (randomly selected) per individual, acquired at 200 × total magnification with a Ziess AxioCam digital camera and Axiovision software (Zeiss, Thornwood, NY). All punctate CD31+ capillaries were manually counted per image and normalized to image area (capillary density).
5
Immunofluorescent Characterization of Bile Duct
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For tissue sections, bile duct was freshly isolated and fixed in 4% paraformaldehyde for 1 hour at room temperature, cryoprotected in 30% sucrose for 3 hours, embedded in optimal cutting temperature (Tissue-Tek; Sakura Finetek), and sectioned at 8 µm. Sections were blocked for 40 minutes at room temperature in blocking solution (5% goat serum or 5% donkey serum, 0.2% Bovine Serum Albumin (BSA), 2% fish gelatin, 0.3% Triton-X, 1X PBS) and incubated with the following primary antibodies: rabbit monoclonal anti-Wt1 (1:200, Abcam, ab89901), rabbit polyclonal anti-Mki67 (1:500, Abcam, ab15580), rabbit polyclonal anti-alpha smooth muscle actin (Acta2) (1:200, Abcam, ab5694), mouse anti-Mesothelin (Msln) (1:200, Santa-Cruz, sc-33672), rat monoclonal anti-Pdgfra (1:200, Thermo, 14-1401-82), rat monoclonal anti-Cd31 (1:200, BD Biosciences, 553369), and rat monoclonal anti-Cd45 (1:200, BD Biosciences, 553076). Secondary Alexa Fluor antibodies (Thermo) were used for detection. For Mki67 and Msln, sections underwent Ag retrieval at 85°C for 15 minutes with Citric Acid pH6 buffer. Vector labs mouse on mouse immunodetection kit (BMK-2202) was used according to the manufacturer’s instructions for Msln.
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