Immunohistochemistry was performed as described previously [17 (link)]. Paraffin wax-embedded tissues were cut into 4 μm sections, mounted on glass slides, and stained using indirect immunoperoxidase. The slides were processed for identification of HIF-1-alpha expression (anti-HIF-1-alpha; Abcam, Cambridge, MA, USA), followed by detection using diaminobenzidine (Sigma Chemical Co., St. Louis, MO, USA). All of the samples were evaluated under an Olympus BX51 microscope. The the areas stained for HIF1-alpha, as percentages of the total area in 10 different fields of each section under 200× magnification, were determined automatically using a digital camera-based image analyzer (Metamorpho, ver. 4.6).
Anti hif 1 alpha
Manufactured by Abcam
Sourced in United Kingdom
Anti-HIF-1-alpha is a lab equipment product that functions as an antibody. It is used for the detection and quantification of the HIF-1-alpha protein in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Diaminobenzidine, by Merck Group (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Ripa buffer, by Wuhan Servicebio Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Wuhan Servicebio Technology (1 mentions)
Anti ifn β, by Abcam (1 mentions)
Ab6940, by Abcam (1 mentions)
Ab82832, by Abcam (1 mentions)
Estrogen receptor alpha, by Abcam (1 mentions)
3 protocols using anti hif 1 alpha
1
Immunohistochemical Analysis of HIF-1-alpha
2
Protein Extraction and Western Blot Analysis
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The cells were lysed in RIPA buffer (Servicebio, China) with a protease inhibitor (PMSF, Servicebio, China) using routine procedures to extract total proteins. Lysates were centrifuged at 12,000 rpm for 10 min, followed by the transfer of supernatants to fresh EP tubes. The BCA protein assay was used for protein quantification. Each sample (25 μg) was loaded onto a 12.5 % SDS-PAGE. The membrane was treated with 5 % non-fat milk for 1 h and then washed three times with TBST (Servicebio, China). After washing, membranes were left overnight at 4 °C with anti–IFN–β (1:1000, Abcam, UK) and anti–HIF–1 alpha (1:1000, Abcam, UK) antibodies. After washing, the membranes were incubated for 2 h with anti-rabbit secondary antibodies. Membranes were developed using enhanced chemiluminescence (ECL).
3
Quantifying ER-alpha and HIF-1alpha Expression
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The expressions of HIF-1alpha and ER-alpha were examined by Western Blot (n = 3). The resulting blots were probed with rabbit monoclonal (#EPR4097, Estrogen Receptor alpha, Abcam, UK), rabbit polyclonal (#ab82832, Anti-HIF-1 alpha, Abcam, UK), and secondary antibodies (#ab6940, Goat polyclonal Secondary Antibody to Rabbit IgG; H&L, Abcam, UK), respectively (Fig. 4, A-1 & A-2). The expressions of ER-alpha (1:200 dilution; ZETA, Bosterbio, USA) for MCF-7CH and MCF-7 were also examined by immuno uorescence assay for three times. Fluorescent gathered in the nucleus of ER positive cells. Photos were taken for more than 100 cells before analyzed by ImageJ (Fig. 4, B-1 & B-2).
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