The extraction of total protein from the bone tissues in the defect areas of the calvaria was performed by using the triplePrep kit (GE Healthcare Life Sciences), and its concentration was determined with a BCA assay kit. 25 μg protein per sample was separated via 10% SDS-PAGE and transferred to the PVDF membrane as described (Luo et al., 2021 (link)). After blocking in 5% skim milk at room temperature for 2 h, PVDF membranes were treated with the following primary antibodies overnight at 4°C, rabbit polyclonal anti-BMP-2 (1:1000, bs-1012R, Bioss), rabbit polyclonal anti-VEGF (1:1000, bs-1313R, Bioss), and rabbit polyclonal anti-PDGF (1:1000, bs-0196R, Bioss), followed by incubation with HRP-labeled IgG at 37°C for 2 h. The western blots were stained with an ECL reagent (Santa Cruz Biotech, CA, United States) and visualized by the ChemiDoc Touch Imaging System—Bio-Rad Laboratories (Bio-Rad Lab, Hercules, CA, United States).
Bs 1313r
Manufactured by Bioss Antibodies
Sourced in United States, China
The Bs-1313R is a laboratory equipment product offered by Bioss Antibodies. It is designed for general laboratory use, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Additional information about the intended use or specific features of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab6672, by Abcam (2 mentions)
Ab78486, by Abcam (2 mentions)
Ab51983, by Abcam (2 mentions)
Ecl reagent, by Santa Cruz Biotechnology (1 mentions)
Bs 0196r, by Bioss Antibodies (1 mentions)
Tripleprep kit, by GE Healthcare (1 mentions)
Bs 1012r, by Bioss Antibodies (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Ab2350, by Abcam (1 mentions)
Digital slice scanner, by 3DHISTECH (1 mentions)
Ar1071, by Boster Bio (1 mentions)
Gtx13418, by GeneTex (1 mentions)
A4923, by ABclonal (1 mentions)
Ab131368, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
Ta 08, by ZSGB-BIO (1 mentions)
Af5135, by Affinity Biosciences (1 mentions)
Readyprep, by GE Healthcare (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Bs 1206r, by Bioss Antibodies (1 mentions)
10 protocols using bs 1313r
1
Protein Extraction and Western Blot Analysis
2
Immunofluorescence Staining of VEGF and Receptors
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IF staining of the cells, the PTCs were made onto slides, firstly fixed with 4% paraformaldehyde solution, and then blocked with goat serum for 20 min. After the blocking process was completed, cells were covered with the primary antibody dilutions, including anti-VEGF (1:50, bs-1313R, Bioss, USA), anti-VEGF-R1 (1:200, ab2350, Abcam, Britain) and anti-VEGF-R2 (1:200, BS1373, Bioworld, USA), and incubated at 37 °C for 60 min, then rinsed in PBS wash buffer. Next, secondary antibody (1:200, Ebiogo, Hefei, China) was added to the cells and cultured at 37 °C for 30 min, then rinsed in the same way. Finally, slides were sealed by ProLong Gold Antifade Reagent with DAPI (Ebiogo, Hefei, China) and scanned using digital slice scanner (3DHISTECH, Hungary).
IF staining of the tissues, the paraffin-embedded decidual tissues, with a thickness of 4μm, were deparaffinized and rehydrated firstly. Then, endogenous peroxidase activity was quenched, and antigen was retrieved in a microwave oven (Midea, China). The following steps are the same as IF staining of the cells. Randomly, select 3 samples from each group for the experiment.
IF staining of the tissues, the paraffin-embedded decidual tissues, with a thickness of 4μm, were deparaffinized and rehydrated firstly. Then, endogenous peroxidase activity was quenched, and antigen was retrieved in a microwave oven (Midea, China). The following steps are the same as IF staining of the cells. Randomly, select 3 samples from each group for the experiment.
3
Histological Analysis of Femoral Head
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After 4 weeks of treatment, the rabbits were anesthetized using intravenous
administration with 3% sodium pentobarbital (30 mg/kg) in an ear vein and the
animals were subsequently sacrificed via air embolism. The right femoral head
was divided into two parts along the coronal plane of the central hole, one
placed in liquid nitrogen cryopreservation and another fixed with 10%
formaldehyde solution (Sangon Biotech Co., Ltd., Shanghai, China) at 4°C for 48
h. Then the femoral head was decalcified with EDTA decalcification solution
(AR1071; Boster Biological Technology, Ltd., Wuhan, China) for 2 months and
embedded in paraffin. Sections of 5 μm thickness were made and stored in
thermostat of 37°C. Specimen sections were stained with hematoxylin and eosin
(HE) and Masson staining.
For immunohistochemical (IHC) staining, sections were deparaffinized, antigen
retrieved, blocked, and incubated with primary antibodies of OCN (1:400;
GTX13418; GeneTex, Irvine, CA, USA) and vascular endothelial growth factor
(VEGF) (1:250; BS1313R; Bioss, Beijing, China) and relevant secondary
antibodies. Then sections were stained with DAB (3,3′-diaminobenzidine) and
counterstained with hematoxylin. At last, each piece of the sections was
observed by light microscope (Olympus Corporation, Tokyo, Japan).
administration with 3% sodium pentobarbital (30 mg/kg) in an ear vein and the
animals were subsequently sacrificed via air embolism. The right femoral head
was divided into two parts along the coronal plane of the central hole, one
placed in liquid nitrogen cryopreservation and another fixed with 10%
formaldehyde solution (Sangon Biotech Co., Ltd., Shanghai, China) at 4°C for 48
h. Then the femoral head was decalcified with EDTA decalcification solution
(AR1071; Boster Biological Technology, Ltd., Wuhan, China) for 2 months and
embedded in paraffin. Sections of 5 μm thickness were made and stored in
thermostat of 37°C. Specimen sections were stained with hematoxylin and eosin
(HE) and Masson staining.
For immunohistochemical (IHC) staining, sections were deparaffinized, antigen
retrieved, blocked, and incubated with primary antibodies of OCN (1:400;
GTX13418; GeneTex, Irvine, CA, USA) and vascular endothelial growth factor
(VEGF) (1:250; BS1313R; Bioss, Beijing, China) and relevant secondary
antibodies. Then sections were stained with DAB (3,3′-diaminobenzidine) and
counterstained with hematoxylin. At last, each piece of the sections was
observed by light microscope (Olympus Corporation, Tokyo, Japan).
4
Parotid Histological Changes After Radiation
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Parotids were collected at 1, 5 or 20 weeks after radiation therapy and fixed in 4% paraformaldehyde for histological or immunohistochemical detection. Samples were dehydrated with gradient ethanol and embedded in paraffin or frozen to obtain sections. Paraffin sections and frozen sections were subjected to immunohistochemistry (IHC) and immunofluorescent (IF) staining, respectively. These sections were incubated overnight at 4 °C with primary antibodies against AQP5 (1:200, ab78486), Ptch1 (1:200, ab51983), IL-6 (1:200, ab6672) (these 3 antibodies are from Abcam, Boston, MA, USA), ARG1 (1:100, A4923, Abclonal, Woburn, MA, USA), or VEGF (1:100, bs-1313R, Bioss, Woburn, USA). After being washed with PBS, appropriate secondary antibodies were incubated for 30 min at room temperature, and nuclei were counter-stained with DAPI for IF staining. The stained sections were imaged and quantified by investigators blinded to the treatments carried out in the experiment. At least two sections from 3 independent parotids were randomly chosen, and 2 random 200× fields of each section were imaged for quantification (n = 2 × 3 × 2 = 12).
5
Protein Extraction and Western Blot Analysis
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The total protein was abstracted by adding the corresponding pyrolysis solution (4°C for 30 minutes) (28–9425–44, ReadyPrep; GE Healthcare Life Sciences). After centrifugation at 876×g for 10 minutes, the supernatant was collected. Protein concentration was determined using bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins were processed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and later transferred onto nitrocellulose membrane as previously described [11 (link)]. After blocking with 5% nonfat milk (with Tween-20) at room temperature for 2 hours, the membrane were incubated with the primary antibodies against GAPDH (1:1000, TA-08, ZSbio, China), VEGF (1:1000, bs-1313R, Bioss), Sirt3 (1:1000, AF5135, Affinity), and LC3 (1:1000, ab48394, Abcam) overnight at 4°C. The secondary antibody (1:10 000, ab131368, Abcam, USA) was incubated with the membrane for 2 hours at room temperature. The expression of target proteins was normalized to GAPDH. At least 6 repeats were included in the western blotting.
6
Comprehensive Molecular Profiling of Parotid Tissue
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Total RNA extraction, reverse transcription, and quantitative PCR (qPCR) were performed as reported [44 (link),45 (link)]. Primers for swine GAPDH, C1qA, TNF-α, IFN-γ, IL-4, IL-6, P53, AIF1, ADGRE 1, ITGAX, HGF, and ARG 1 were designed using Primer3 software (http://bioinfo.ut.ee/primer3/ ) and listed in Supplementary Table S1 . For protein extraction, fresh parotid samples were homogenized in T-PER reagent containing protease inhibitors (Pierce, WA, USA). The concentration of protein was quantified using BCA, and the loading quantity was 25 μg per lane. Western blotting was done using primary antibodies against AQP5 (1:5000, ab78486), Ptch1 (1:5000, ab51983), TNF-α (1:5000, ab1793), IL-6 (1:200, ab6672), P53 (1:500, ab154036) (these 5 antibodies are from Abcam, Boston, MA, USA), Gli1 (1:1000, bs-1206R), F4/80 (1:1000, bs-7058R), AIF1 (1:1000, bs-1363R), and VEGF (1:100, bs-1313R) (these 4 antibodies are from Bioss, Woburn, MA, USA). All PCR and Western blot analyses were repeated at least three times.
7
Western Blot Analysis of Callus Proteins
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The proteins from the callus tissues were extracted by using lysis buffer (Beyotime, Shanghai, China). Then the protein was quantified by the BCA assay kit (Beyotime, Shanghai, China). After that, an equal amount of proteins was electrophoresed on SDS-PAGE and then transferred onto PVDF membranes (Thermo Fisher, Cambridge, MA, USA). Next, the membranes were immuno-blotted with Hypoxia-inducible factor-1α (HIF-1α) rabbit antibody (1: 500 dilution, Catalog No.: AF1009, Affinity, Changzhou, Jiangsu, China), vascular endothelial growth factor (VEGF) rabbit antibody (1: 500 dilution, Catalog No.: bs-1313R, Bioss, Beijing, China), Runx2 rabbit antibody (1: 500 dilution, Catalog No.: AF5186, Affinity), Osterix (1: 400 dilution, Catalog No.: DF7731, Affinity), Type I collagen antibody (1: 1000 dilution, Catalog No.: AF7001, Affinity), SDF-1α rabbit antibody (1: 500 dilution, Catalog No.: AF5166, Affinity), CXCR4 antibody (1: 1000 dilution, Catalog No.: A12534, ABclonal, Wuhan, China), or β-actin mouse antibody (1: 2000 dilution, Catalog No.:60008-1-Ig, proteintech, Wuhan, Hubei, China). The membranes were washed three times and incubated with goat HRP-conjugated secondary antibodies (proteintech, Wuhan, Hubei, China). The specific bands were developed with the ECL system (7 Sea biotech, Shanghai, China). β-actin was used as the control of HIF-1α and VEGF.
8
Western Blot Quantification of VEGFA
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Protein extraction was performed using a RIPA buffer supplemented with protease inhibitor. The protein concentration was determined using a BCA assay kit (Thermo Scientific, USA). Protein samples were separated by 12% SDS-PAGE and then transferred onto PVDF membranes (Millipore, USA). The membranes were stained using an enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate (Bioworld Technology, China) according to the manufacturer's instructions. Images were acquired using a GBOX ChemiXRQ System (Syngene, UK), and protein expression levels were quantified with ImageJ software. The main reagents used included rabbit anti-VEGFA polyclonal antibody (bs-1313R, Bioss), anti-GAPDH antibody (Proteintech, China), ECL (K-12043-D10, China), TRIS (T8060, Solarbio), HRP-labeled goat anti-rabbit IgG (SA00001-2, Proteintech), and HRP-labeled goat anti-mouse IgG (SA00001-1, Proteintech).
9
Diabetic Rat Skin Tissue Analysis
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The diabetic rats were euthanized, and skin tissues were removed from the full‐thickness skin defects and burns of the rats. The tissues were fixed in neutral formaldehyde for 1 d. Then, the tissues had been embedded in paraffin and sectioned before being subjected to H&E, DHE, and immunofluorescent staining. Immunofluorescence single staining: VEGF (bs‐1313R, Bioss), CD86 (13395‐1‐AP, Proteintech), CD206 (60143‐1, Proteintech), TNF‐α (bs‐10802R, Bioss), HIF‐1α (bs‐1407R, Bioss), MMP‐9 (bs‐4593R, Bioss), IL‐6 (bs‐0782R, Bioss) and IL‐10 (bs‐6761R, Bioss). Immunofluorescence double staining: α‐SMA (bsm‐33188 M, Bioss)/CD31 (GB11063‐2, Servicebio) and Col I (14695‐1‐AP, Proteintech)/Vimentin (GB12192, Servicebio)). The cell nucleus of the tissues were labeled with DAPI, and the photographs were analyzed for intensities of fluorescent signals using ImageJ software. The intensities of the fluorescent signals in the full‐thickness skin defects and burns of the diabetic rats in the control groups were set to 100%. Laser Speckle Contrast Imaging (RFLSI Pro) was used to examine blood perfusion in the full thickness skin defects and burns.
10
Collagen and HDAC2 Regulation in Angiogenesis
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Erythromycin enteric-coated tablets (H42021990, Yichang Humanwell Pharmaceutical Co., LTD.); Vorinostat Capsules (180509, Beijing Hengrui Kangda Medical Science and Technology Development Co., Ltd.); Budesonide (AstraZeneca 8339000); Rabbit Anti-Collagen III Polyclonal Antibody (bs-10423R, Bioss); Rabbit Anti-Collagen I Polyclonal Antibody (bs-0549R, Bioss); Rabbit Anti-HDAC2 Polyclonal Antibody (bs-1813R, Bioss); Rabbit VEGF ELISA kit (MM-021001); Rabbit TGF-β1 ELISA kit (MM-3684001); Rabbit Polyclonal Anti-VEGF (bs-1313R, Bioss, 1/500–1/2000); Rabbit Polyclonal Anti-TGFβ1 (bs-0086R, Bioss, 1/500–1/2000); Rabbit monoclonal Anti-IL-8 (ab34100, abcam, 1/1000); Rabbit Polyclonal Anti-HDAC2 (OmnimAbs, OM105905, 1/500–1/2000); fluorescence microscope (CKX53, OLYMPUS); Microplate Reader (RT-6100, Rayto); Protein vertical electrophoresis instrument (DYY-6C, Beijing 61 instrument factory); Ultra High Sensitivity Chemiluminescence Imaging System (Chemi DocTM XRS+, Bio-Rad Shanhhai Laboratories).
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