The expression of Collagen IV, Laminin gamma3 and Fibrillin 2 within ovarian tissues were detected by immunohistochemistry staining on the paraffin slide respectively. The ovarian sections fixed on the paraffin performed dewaxing and rehydration, antigen retrieval, and incubated in wet box with 3% H2O2 for 10 min sequentially. After blocking with 3% BSA, the slides were then immunostained with primary antibodies of Collagen IV (1:400, Abcam, ab236640), Laminin gamma3 (1:200, Abcam, ab234429) and Fibrillin 2 (1:200, Proteintech, 20252-1-AP) in a humidified box at 4 ℃ overnight. The slides were washed three times, and incubated with secondary antibody anti-rabbit (1:500, Sangon Biotech, D110065) at 37 °C for 30 min. Then the slides were stained with diaminobenzidine (DAB) as chromogen and counterstained with hematoxylin according to manufacturer’s instruction. After dehydration, the coverslips were mounted, and immunohistochemical images were taken using Leica Camera (Leica Microsystem, Buffalo Grove, IL, United States). Protein expressions were quantified with Image J software.
D110065
Manufactured by Sangon
The D110065 is a laboratory centrifuge capable of separating mixtures of liquids, solids, and gases based on differences in their densities. It is designed to operate at high speeds to generate the necessary centrifugal force for this purpose.
Automatically generated - may contain errors
2 protocols using d110065
1
Immunohistochemical Analysis of Ovarian Proteins
2
Quantitative Western Blot Analysis
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Total protein in cells or xenograft tumor tissues (see details below) was extracted using the mixture of cell lysis buffer and protease inhibitor. The protein sample was separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with non-fat milk and then probed with the primary antibodies including NRIP1 (1:500, A9955, ABclonal Technology Co., Ltd., Wuhan, Hubei, China), B-cell lymphoma-2 (Bcl-2; 1:500, A0208, ABclonal), Bcl-2-associated X (Bax; 1:500, A0207, ABclonal), NSD2 (1:500, GTX106306, GeneTex Inc., San Antonio, TX, USA), DGCR8 (1:1,000, ab191875, Abcam Inc., Cambridge, MA, USA), H3K36me2 (1:2,000, ab176921, Abcam), and β-actin (1:500, GTX109639, GeneTex) overnight at 4°C. Thereafter, the membranes were further incubated with HRP-conjugated mouse anti-rabbit IgG (1:2,000, D110065, Sangon Biotech) at room temperature for 1 h. The blot bands were developed by enhanced chemiluminescence reagent. The gray value of the protein bands was evaluated by Image J to analyze expression of target proteins.
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