As we described previously, the 20-μm-thick brain slice was fixed with 4% paraformaldehyde (PFA) for further analysis (Shen et al., 2018 (link)). In brief, blocking buffer (0.3% Triton X-100 and 10% normal goat serum) was applied for 2 h to block nonspecific binding and primary antibody of HIF-1α (1:100; Novus), VEGF (1:100; Abcam), Dll4 (1:50; Abcam), RECA-1 (1:50; Abcam), Notch (1:50; Cell Signaling Technology), CC1 (1:100; Abcam), and MBP (1:200; Cell Signaling Technology) were incubated overnight at 4°C. Sections were incubated with 488- (1:800, Life Technology) or Cy3-conjugated (1:800, KPL) secondary antibody for 2 h at room temperature. Confocal images were obtained using a laser scanning confocal microscope (Zeiss LSM 700, Carl Zeiss).
Reca 1
Manufactured by Abcam
Sourced in United States, United Kingdom
RECA-1 is a recombinant protein that functions as a DNA repair enzyme. It is involved in the homologous recombination pathway, which is responsible for the repair of double-strand breaks in DNA. The core function of RECA-1 is to facilitate the strand exchange and strand invasion steps of the homologous recombination process.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Hif 1α, by Novus Biologicals (1 mentions)
Anti mouse igg alexafluor 488, by Thermo Fisher Scientific (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Prolong gold antifade mountant with 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Anti nf 160, by Merck Group (1 mentions)
Axio observer d1 microscope, by Zeiss (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Cy3 conjugated anti gfap, by Merck Group (1 mentions)
Cyp2e1, by Abcam (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti α sma, by Merck Group (1 mentions)
Click it reaction cocktail, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Solarbio (1 mentions)
Hif 1α, by Abcam (1 mentions)
Lsm 700 confocal laser scanning microscope, by Zeiss (1 mentions)
Versican, by Merck Group (1 mentions)
12 protocols using reca 1
1
Immunofluorescence Analysis of Brain Tissue
2
Visualizing Ovalbumin-Alexa Fluor 647 in Blood Vessels
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For visualisation of ovalbumin-Alexa Fluor 647 sections were imaged on a confocal laser scanning microscope (LSM880, Zeiss). To determine the location of tracer with regards to the walls of blood vessels, sections were incubated with Rat Endothelial Cell Antibody (1:100; RECA-1; AB9774 Abcam, Cambridge, United Kingdom) followed by a secondary antibody (1:400; anti-mouse IgG Alexa Fluor 488; Molecular Probes, Life Technologies, New York, USA) and with actin α-smooth muscle antibody (1:400; SMA-Cy3; Sigma-Aldrich, St. Louis, Montana). Images were acquired in a z-stack scan mode, with interval of 0.5 μm, as a 4 line average, pixel dwell 0.76 μs, using a plan-apochromat 40x/1.3 oil DIC UV-IR M27 objective. Image dimensions were x: 1024, y: 1024, z: 36 μm; 8-bit.
3
Occludin and RECA-1 Co-localization
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Co-immunostaining was performed on 4% paraformaldehyde fixed 16-μm-thick brain slices to colocalize the expression of occludin (antibody dilution 1:50, Life technologies) and rat endothelial cell antigen-1 (RECA-1, antibody dilution 1:30, Abcam, USA). Images were captured on a fluorescence microscope (Olympus IX71).
4
Multicolor Immunofluorescence Staining of Liver, Spleen, Kidney, and Lung Tissues
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A section of the left lobe of the liver, half of the spleen, one kidney, and one lung were cut into 4 × 4 mm pieces and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 48 h, and then transferred to 70% ethanol. Following dehydration, samples were embedded in paraffin (Histolab Products AB). After rehydration and antigen retrieval, tissue sections were incubated overnight with a rabbit antiserum against HBP at a 1:3,000 dilution and one of the following antibodies: monoclonal mouse anti-rat endothelial cell antigen 1 (RECA-1; Abcam; clone RECA-1; a pan-endothelial cell marker), monoclonal mouse anti-rat CD68 (Abcam; clone ED1; a pan-monocyte/macrophage marker), and monoclonal mouse anti-rat asialoglycoprotein receptor (ASGR1; Invitrogen; clone 8D7; a hepatocyte marker) at a 1:100 dilution. HBP antiserum from rabbit was provided by Heiko Herwald, originally obtained as described previously [28 (link)]. Samples were then stained with secondary Alexa Fluor-647-conjugated goat anti-rabbit and Alexa Fluor-568-conjugated goat anti-mouse Fab2′ antibody fragments. Coverslips (Menzel-glaser; #1.5 thickness) were mounted on the samples using ProLong Gold Antifade Mountant with 4,6-diamidino-2-phenylindole (Life Technologies).
5
Spinal Cord Injury Histological Analysis
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The animals were sacrificed 3 months after hydrogel implantation. They were then deeply anesthetized with an intraperitoneal injection of overdose pentobarbital and perfused with physiological saline, followed by 4% paraformaldehyde in 0.1M phosphate buffer. The spinal cord was left in the bone overnight, then removed and postfixed in the same fixative for at least 1 week.
A 4 cm-long segment of the spinal cord with the lesion site in the middle was dissected, and a series of 40 mm-thick longitudinal sections were collected. Hematoxylin–eosin staining was performed, using standard protocols, and the slides were specifically evaluated using an Axio Observer D1 microscope (Carl Zeiss Microimaging GmbH, Oberkochen, Germany). For immunohistochemical studies, the following primary antibodies and dilutions were used: Cy3-conjugated anti-GFAP (1:200; Sigma-Aldrich, Saint Louis, MO, USA) to identify astrocytes, anti-NF 160 (1:200; Sigma-Aldrich, Saint Louis, MO, USA) to identify neurofilaments, and RECA-1 (1:50; Abcam, Cambridge, UK) to identify endothelial cells of blood vessels. Alexa Fluor 594 goat anti–rabbit IgG (1:200; Invitrogen) and Cy3-conjugated anti-mouse IgM (1:100; Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies.
A 4 cm-long segment of the spinal cord with the lesion site in the middle was dissected, and a series of 40 mm-thick longitudinal sections were collected. Hematoxylin–eosin staining was performed, using standard protocols, and the slides were specifically evaluated using an Axio Observer D1 microscope (Carl Zeiss Microimaging GmbH, Oberkochen, Germany). For immunohistochemical studies, the following primary antibodies and dilutions were used: Cy3-conjugated anti-GFAP (1:200; Sigma-Aldrich, Saint Louis, MO, USA) to identify astrocytes, anti-NF 160 (1:200; Sigma-Aldrich, Saint Louis, MO, USA) to identify neurofilaments, and RECA-1 (1:50; Abcam, Cambridge, UK) to identify endothelial cells of blood vessels. Alexa Fluor 594 goat anti–rabbit IgG (1:200; Invitrogen) and Cy3-conjugated anti-mouse IgM (1:100; Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies.
6
Immunofluorescence Characterization of Hepatocytes and Cholangiocytes
7
Penile Tissue Immunohistochemical Staining
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Penile mid-shaft tissues were harvested, fixed and further processed for immunohistochemical staining as previously described [15 (link)]. Briefly, tissues were fixed in cold 2% formaldehyde and 0.002% picric acid in 0.1 M phosphate buffer, pH 8.0, for 4 hours followed by overnight immersion in buffer containing 30% sucrose. The specimens were then embedded in OCT Compound (American Master Tech Scientific, Lodi, CA, USA) and stored at -70°C until use. Sections were cut at 5 μm, mounted into charged slides and air dried for 5 minutes. Representative slides were stained with Masson’s trichrome for connective tissue and smooth muscle histology.
For immunohistochemical examination, tissue sections were stained with mouse anti-rat endothelial cell antigen-1 (RECA-1, Abcam Inc, Cambridge, MA, USA) and terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL, Roche Diagnostics Corporation, Indianapolis, IN, USA) using standard techniques [16 (link)]. To visualize ADSC, EdU staining with immunostaining for α-smooth muscle actin (α-SMA) was done with mouse anti-α-SMA (Sigma-Aldrich, St. Louis, MO, USA) and freshly made Click-IT reaction cocktail (Invitrogen, Carlsbad, CA, USA). Nuclear staining was performed with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) [26 (link)].
For immunohistochemical examination, tissue sections were stained with mouse anti-rat endothelial cell antigen-1 (RECA-1, Abcam Inc, Cambridge, MA, USA) and terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL, Roche Diagnostics Corporation, Indianapolis, IN, USA) using standard techniques [16 (link)]. To visualize ADSC, EdU staining with immunostaining for α-smooth muscle actin (α-SMA) was done with mouse anti-α-SMA (Sigma-Aldrich, St. Louis, MO, USA) and freshly made Click-IT reaction cocktail (Invitrogen, Carlsbad, CA, USA). Nuclear staining was performed with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) [26 (link)].
8
Immunofluorescence Staining of HIF-1α and RECA-1 in Cerebral Ischemia
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The rats were immediately perfused with PBS and 4% PFA after cerebral ischemia for 2 hours. We followed the previously published methods of Shen et al. in doing immunofluorescence staining [13 (link)]. In brief, 20 μm thick cryosections were preincubated for 1 hour at room temperature in PBS which contained 0.1% Triton X-100, 1% BSA, and 5% goat serum (Solarbio, Beijing, China) to cover nonspecific binding sites. The HIF-1α (1 : 200, Abcam) and RECA-1 (1 : 100, Abcam) primary antibodies were applied to the brain slices and incubated overnight at 4°C. Appropriate secondary antibodies that bind to Cy3 (anti-mouse, 1 : 800) or 488 (anti-rabbit, 1 : 800) were used for detection. The nucleus was stained with Dapi. The LSM 700 confocal laser scanning microscope (Zeiss) was used to take images from the ischemic area and the mirror nonischemic area [13 (link)].
9
Immunohistochemical Staining of Penile Tissues
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Freshly dissected penile tissues were harvested, fixed, and processed for immunohistochemical staining, as previously described.6 (link),7 (link),13 (link), 14 (link), 15 (link) After fixation penile tissues were embedded in OCT Compound (American Master Tech Scientific, Lodi, CA, USA), and 5-μm thick section were cut and mounted on glass slides. The sections were then processed for immunohistochemical staining with mouse antineuronal nitric oxide synthase (nNOS, 1:100; BD Biosciences, San Jose, CA, USA), mouse anti-rat endothelial cell antigen-1 (1:400, RECA-1; Abcam, Cambridge, MA, USA), phalloidin (1:400; Invitrogen, Carlsbad, CA, USA), and mouse anti-8-hydroxy-2′-deoxyguanosine (8-OHdG, 1:50, JaICA, Nikken Seil Co, Japan) by using standard techniques. Cell apoptosis was assessed using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL, Roche Diagnostics Corporation, Indianapolis, IN, USA) kit. Briefly, the slides were incubated in methanol/hydrogen peroxide for 5 minutes to quench endogenous peroxidase activity. Subsequently, the specimens were incubated in TUNEL reagent (1 hour at 37°C) and anti-digoxigenin-peroxidase (30 minutes at room temperature). Finally, nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA), followed by counterstaining with fluorescence mounting medium.
10
Tissue Fixation, Sectioning, and Staining for Histological Analysis
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After euthanasia, tissue samples were fixed in cold 2% formaldehyde and 0.002% picric acid in 0.1 M phosphate buffer, pH 8.0, for 4 hours followed by overnight immersion in buffer containing 30% sucrose. The specimens were then embedded in OCT Compound (American Master Tech Scientific, Lodi, CA, USA) and stored at -70°C until use. Sections were cut at 5 μm, mounted into charged slides and air dried for 5 minutes. Representative slides were stained with Masson’s trichrome for connective tissue and smooth muscle histology.
For immunohistochemical examination, tissue sections were stained with mouse anti-rat endothelial cell antigen-1 (RECA-1, Abcam Inc, Cambridge, MA, USA) and terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL, Roche Diagnostics Corporation, Indianapolis, IN, USA) using standard techniques [15 (link)].
For immunohistochemical examination, tissue sections were stained with mouse anti-rat endothelial cell antigen-1 (RECA-1, Abcam Inc, Cambridge, MA, USA) and terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL, Roche Diagnostics Corporation, Indianapolis, IN, USA) using standard techniques [15 (link)].
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