Six sections of the middle area of the flap from each group were deparaffinized in xylene, and then, graded ethanol baths were used for rehydration. After rehydrating, the specimens were blocked with 3% (v/v) hydrogen peroxide. Subsequently, 10.2 mM sodium citrate was applied for antigen repair for 30 min at 95°C. Then, the following primary antibodies were used for incubation overnight as 4°C, including CD34 (1 : 100, ab81289, Abcam), Cadherin5 (1 : 100, A02632-2, Boster Biological Technology), VEGF (1 : 300, A12303, ABclonal), CASP3 (1 : 200, 19677-1-AP, Proteintech), SOD1 (1 : 100, 10269-1-AP, Proteintech), and CTSD (1 : 100, 21327-1-AP, Proteintech). Then, samples were treated with HRP-conjugated secondary antibody (SA00001-1, Proteintech), stained by DAB kit, and counterstained with hematoxylin. Lastly, stained sections were visualized under light microscopy using the DP2-TWAN image-acquisition system (Olympus Corp., Tokyo, Japan). Quantification was performed for Cadherin 5, VEGF, SOD1, CASP3, and CTSD expression levels, and the number of CD34-positive blood vessels was enumerated. Measurements were obtained from three random sections, with six random visual fields each.
A12303
Manufactured by ABclonal
Sourced in China
A12303 is a pipette designed for accurate and precise liquid handling. It features an adjustable volume range and is suitable for a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab81289, by Abcam (1 mentions)
Dp2 twan image acquisition system, by Olympus (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Ab144422, by Abcam (1 mentions)
Ab182981, by Abcam (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Ab138492, by Abcam (1 mentions)
Testicular hyaluronidase, by Merck Group (1 mentions)
Ab93876, by Abcam (1 mentions)
Hydrogen peroxide solution, by Merck Group (1 mentions)
3 3 diaminobenzidine dab solution, by Vector Laboratories (1 mentions)
A0378, by ABclonal (1 mentions)
Image lab analysis software, by Bio-Rad (1 mentions)
Ab197493, by Abcam (1 mentions)
Yt5845, by Immunoway (1 mentions)
Ac026, by ABclonal (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Goat serum, by ABclonal (1 mentions)
Methanol, by Merck Group (1 mentions)
Eclipse ti2, by Nikon (1 mentions)
7 protocols using a12303
1
Immunohistochemical Assessment of Angiogenic Factors
2
Evaluation of Angiogenesis Regulators
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3
Protein Extraction and Western Blot Analysis
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In order to obtain protein samples, the retinas or HRCECs were placed in RIPA buffer with a protease inhibitor (phenylmethanesulfonyl fluoride), crushed using an ultrasonic cell pulverizer, centrifuged (5,000 × g/2 min at room temperature) to obtain the supernatant protein, and quantified using a BCA Protein Assay kit (Nanjing KeyGen Biotech. Co., Ltd.). Equivalent amounts of protein (20 µg) from each sample were run at 10% SDS-PAGE and transferred to 0.22 µm polyvinylidene fluoride membranes (EMD Millipore). After blocking with 5% non-fat dried milk solution at room temperature for 2 h, the membranes were first incubated overnight at 4°C with polyclonal rabbit anti-vascular endothelial growth factor (VEGF) antibody (A12303, ABclonal; 1:3,000), polyclonal rabbit anti-pigment epithelium-derived factor (PEDF) antibody (A11782, ABclonal; 1:3,000), poly-clonal rabbit anti-PLK4 antibody (12952-1-AP, ProteinTech Group, Inc.; 1:1,000) or monoclonal mouse anti-β-tubulin antibody (CW0098, ComWin Biotech; 1:5,000), and then with appropriate peroxidase-labeled secondary anti-rabbit or anti-mouse antibodies (RM3002, or RM3001, Beijing Ray Antibody Biotech; 1:3,000) at room temperature for 1 h. Chemiluminescent signals were visualized using Ncm-ECL Ultra (New Cell & Molecular Biotech Co., Ltd.), while ImageJ software was used to determine the gray strip values.
4
Immunohistochemical Staining of Bone Markers
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For IHC staining, the tissue sections were subjected to deparaffinization using xylene, followed by hydration using graded ethanol. To inhibit endogenous peroxidase activity, the sections were then incubated in a 3 % hydrogen peroxide solution (Sigma-Aldrich). Additionally, the sections were treated with 2 mg/mL of testicular hyaluronidase (Sigma-Aldrich) for 30 min at 37 °C. Subsequently, the sections were blocked using 1.5 % goat serum and incubated with specific primary antibodies overnight at 4 °C, including anti-COL1A1 (1:500, ab138492, Abcam), anti-osteocalcin (1:500, ab93876, Abcam), anti-CD31 (1:500, A0378, ABclonal, Wuhan, China), and anti-VEGFA (1:500, A12303, ABclonal). The following day, the sections were incubated with biotinylated goat anti-rabbit secondary antibodies for a duration of 30 min. Signal amplification was achieved using the Vectastain Elite ABC kit. The staining process involved the use of a 3,3′-diaminobenzidine (DAB) solution (all from Vector Laboratories, Burlingame, CA, USA), and counterstaining of the nuclei was performed using hematoxylin. Images of 5 randomly chosen fields in each group were taken with a bright field microscope. The Image J software (NIH, Bethesda, MD, USA) was used to analyze the average gray values to obtain immunohistochemical staining quantitative results.
5
Quantifying Ischemic Brain Protein Markers
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Ischemic brain tissue (cell protein) extract was assayed with a BCA kit according to the manufacturer’s protocols towards obtaining accurate concentration of proteins and then added with 1/4 volume of protein buffer and denatured by heating. Aliquots of the samples (40 μg) were analysed through SDS-PAGE gel. Following this, the gel was transferred and then blocked and incubated with different corresponding primary antibodies against HIF-1α (ab197493, Abcam, Danvers, MA), VEGF (A12303, ABclonal, Wuhan, China), VEGFR2 (YT5845, Immunoway, Plano, TX), PLCγ1 (AF8390, Affinity, San Francisco, CA), PKC (YT3752, Immunoway, Plano, TX), eNOS (CPA9156, Bejing, China) overnight. Subsequently, the target membranes were incubated with a secondary antibody. Proteins were detected using the ECL kit and the results were analysed using Image Lab analysis software (Bio-Rad Laboratories, Inc., Hercules, CA).
6
Western Blot Analysis of Angiogenic Factors
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HepG2 and MHCC97H cells treated with or without MTE for 24 h were lysed with RIPA buffer (Beyotime Biotechnology), and protein concentrations were quantified by the BCA method. Proteins were separated by SDS-PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes. After blocking, the membranes were incubated with the primary antibodies against VEGFA (A12303, ABclonal, China), PDGFRB (A2180, ABclonal, China), VWF (A13523, ABclonal, China), and the internal reference β-actin (AC026, ABclonal, China) at 4°C overnight. Also, the membrane was then incubated with the secondary antibody for 1 h at room temperature. After being incubated with the ECL-enhanced luminescent substrate, protein expression was detected using a chemiluminescence image analysis system (Tanon-5200, Shanghai, China).
7
Immunofluorescent Analysis of VEGFA Expression
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HUVECs were cultured with material samples for 3 days and washed twice with PBS. HUVECs were fixed with methanol (Merck, USA) in −20 °C for 15 min and then permeated in 0.1%Triton-x100 in 5 % goat serum (Solarbio) for 5 min at room temperature. Primary antibody of VEGFA rabbit pAb (A12303, Abclonal, China) was diluted at 1:100 in 5 % goat serum and incubated on HUVECs at 4 °C overnight. Then HUVECs were stained with a secondary antibody of FITC Goat anti-Rabbit IgG (H + L) (AS011, Abclonal, China) at 1:100 in 5 % goat serum for 1 h at room temperature. DAPI was stained for nuclear imaging. Immunofluorescent images were observed and captured using a laser scanning confocal microscope (Nikon Eclipse Ti2, Japan). The mean intensity was counted in 5 random fields and analyzed using Image J software.
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