IHC staining of the PCa Donor Rapid Autopsy Program at the University of Washington (UWRA, Seattle, WA) metastatic CRPC tissue microarray was performed using SR-B1 primary antibody: AB52629 (Abcam, Cambridge, United Kingdom) (27 (link)). Metastatic specimens were obtained from patients who died of metastatic CRPC, who signed written informed consent for a rapid autopsy performed within 6 h of death under the aegis of the PCa Donor Program at the University of Washington with Institutional Review Board approval. SR-B1 staining was scored by experienced independent pathologists (0 = no staining, 1 = low staining, 2 = moderate staining, 3 = high staining). Expression data for cholesterol metabolism gene transcripts was obtained from 27 patients with paired normal prostatic, and local cancerous, tissue from the Shanghai Changhai Hospital and Fudan University Shanghai Cancer Center (Shanghai Cohort, SC) (28 ) and from 83 CRPC patients from the UWRA; an expansion of the 63 CRPC patient data previously reported (27 (link)).
Ab52629
Manufactured by Abcam
Sourced in United Kingdom
Ab52629 is a lab equipment product offered by Abcam. It serves as a core component for various laboratory applications. The detailed specifications and intended use of this product are not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab52818, by Abcam (2 mentions)
Ab18180, by Abcam (2 mentions)
Ab52617, by Abcam (2 mentions)
Ab64264, by Abcam (1 mentions)
Sm2000r, by Leica (1 mentions)
Tp1020 automatic tissue, by Leica (1 mentions)
Eg1150c, by Leica (1 mentions)
Fsx100 microscope, by Olympus (1 mentions)
Ab65596, by Abcam (1 mentions)
Ab121000, by Abcam (1 mentions)
Bs 5013r, by Bioss Antibodies (1 mentions)
Ab186429, by Abcam (1 mentions)
Ab63983, by Abcam (1 mentions)
Ab33470, by Abcam (1 mentions)
Sc 2005, by Santa Cruz Biotechnology (1 mentions)
Sc 2020, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Sc 2004, by Santa Cruz Biotechnology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Gradient polyacrylamide gel, by ATTO Corporation (1 mentions)
12 protocols using ab52629
1
Immunohistochemical Analysis of Metastatic CRPC
2
Immunohistochemical Localization of SR-BI and LDL-R in Liver
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The location of SR-BI and low-density lipoprotein LDL-receptor (LDL-R) in liver tissue was evaluated via immunohistochemistry. In brief, liver specimens fixed in 10% formalin at room temperature for 1 week, were embedded in paraffin wax, cut into 5-µm-thick sections, deparaffinized in xylene and rehydrated in a reverse-gradient series of ethyl alcohol. Following treatment with 3% hydrogen peroxide for 15 min at room temperature to block endogenous peroxidase activity, the sections were incubated with primary antibodies against SR-BI (1:100; ab52629; Abcam, Cambridge, UK) and LDL-R (1:100; sc-11824; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 12 h at 4°C then washed with PBS. Biotinylated goat anti-polyvalent secondary antibodies [1:200; Mouse and Rabbit Specific HRP/DAB (ABC) Detection IHC kit; ab64264; Abcam] were added and incubated for 20 min at room temperature. The specimens were subsequently incubated with streptavidin peroxidase for 10 min at room temperature and DAB (1:50) was applied to visualize the labeling. The positive area was identified with brown staining under the B5-223IEP light microscope (magnification, −400).
3
Immunofluorescence Analysis of SR-B1 in Formalin-Fixed Liver
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Formalin-fixed liver was dehydrated in Leica TP1020 automatic tissue hydroextractor (Leica, Germany). Then, tissues were paraffin embedded (Leica EG1150C, Leica, Germany) and sectioned (Leica SM 2000R, Leica, Germany). Liver sections were stained with hematoxylin and eosin or subjected to immunofluorescence. For immunofluorescence staining, tissue sections were deparaffinized and blocked for 40 min in 5% goat serum at room temperature. Then, they were incubated overnight at 4 °C with the primary SR-B1 antibodies (ab52629, Abcam) diluted with 2% goat serum. The sections were rinsed three times in PBST for 5 min each and incubated with anti-rabbit secondary antibodies (Life) for 1 h at RT. Then, the sections were stained with DAPI and mounted onto glass slides after washed in PBST for three times. Photomicrographs were taken using an Olympus FSX100 microscope (X200 original magnification).
4
Protein Expression Analysis in Liver and Intestine
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About 40 mg of frozen liver and the entire middle segment of small intestine that was divided into 5 equal segments (~40 mg) were homogenized in RIPA buffer (50 mMTris-HCl, pH 8.0, 150 mMNaCl, 2 mM MgCl2, 0.1% SDS, 1.5% Nonidet P-40, and 0.5% deoxycholate) with protease inhibitors. The homogenate was centrifuged at 12,000 rpm for 20 min at 4 oC. The total protein concentration of each sample was determined by a BCA protein assay kit (Pierce, Rockford, USA). From each sample, a total of 50 μg of total proteins was subjected to SDS-PAGE (8% for NPC1L1, 6% for ABCA1 and 10% for others) and Western blotting. Primary antibodies included a rabbit anti-mouse CYP7A1 antibody (Cat.#: ab65596, Abcam, UK), a rabbit anti-mouse/human LDL receptor antibody (Cat.#: ab52818, Abcam, UK), a mouse anti-ABCA1 antibody (Cat.#: ab18180, Abcam, UK), a rabbit anti-SR-BI antibody (Cat.#: ab52629, Abcam, UK), a rabbit anti-ABCG5 antibody (Cat.#: BS5013R, Bioss, China), and a goat anti-NPC1L1 antibody (Cat.#: ab121000, Abcam, UK).
5
Immunodetection of Cell Surface Markers
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Rabbit anti-CD36 polyclonal IgG (Proteintech) (1:500 dilution), rabbit anti SR-B1 monoclonal IgG (ab52629, Abcam, Cambridge, United Kingdom) (1:2000 dilution), rabbit anti-CD41 polyclonal Ab (ab63983, Abcam) (1:500 dilution), rabbit monoclonal anti-ALIX (ab186429, Abcam) (1:1,000 dilution), rabbit anti-Apo A I polyclonal Ab (reactive to both mouse and human Apo AI, ab33470, Abcam) (concentration, 1.5 μg/ml) and rabbit anti-CD31 or PECAM-1 polyclonal IgG (Proteintech Group Inc., Rosemont, IL) (1:500 dilution) were used in this study.
6
Western Blot Analysis of Mouse Liver and Serum
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Mouse serum (4 μl) was diluted with 12 μl of PBS, and mouse liver (50 mg) was homogenated using 125 μl of homogenate buffer (20 mM Tris-HCl (pH 7.4), 0.1% SDS, 0.1% Triton X-100, 0.01% sodium deoxycholate and 1 × Complete protease inhibitor cocktail (Roche Diagnostics)). Samples (16 μl) were mixed with 4 μl of Laemmli sample buffer (Bio-Rad, Hercules, CA) and then denatured at 95 °C for 2 min. Total proteins were separated by electrophoresis on a 5–20% gradient polyacrylamide gel (ATTO Corporation, Tokyo, Japan) and transferred onto polyvinylidene difluoride membranes. Blots were probed with goat primary antibodies against Apolipoprotein A-I (1:500, sc-23605, Santa Cruz Biotechnology, Santa Cruz, CA) and Apolipoprotein B (1:500, sc-11795, Santa Cruz Biotechnology), or Scavenging receptor SRB1 (1:2,000, ab52629, Abcam, Cambridge, UK) and β-actin (1:500, ab6276, Abcam), and then incubated with anti-goat secondary antibody (1:2,000, sc-2020, Santa Cruz Biotechnology), anti-rabbit secondary antibody (1:2,000, sc-2004, Santa Cruz Biotechnology) or anti-mouse secondary antibody (1:2,000, sc-2005, Santa Cruz Biotechnology) conjugated with horseradish peroxidase. Blots were visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA) and analysed by a ChemiDoc System (Bio-Rad).
7
Protein Expression and Regulation Analysis
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Total protein was extracted and protein concentration was detected by BCA assay. Western blot analysis was performed as previously described [39 (link)]. EFNB2 (ab69858, Abcam, Cambridge, UK), EPHB4 (ab150545, ab98933, Abcam), LDLR (ab52818, Abcam), β-catenin (ab32572, Abcam), VLDLR (ab203271, Abcam), SCARB1(ab52629, Abcam), STAT3(ab68153, Abcam), p-STAT3(ab267373, Abcam), JAK2(ab108596, Abcam), p-JAK2(ab108596, Abcam), SREBP2(ab30682, Abcam), proliferating cell nuclear antigen (PCNA; Proteintech Group, Inc., Sankt Leon-Rot, Germany) primary antibodies were used. Horseradish peroxidase (HRP)-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00001-2) and HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (SA00001-1) were obtained from Proteintech Group, Inc (Chicago, US).
8
Immunohistochemical Analysis of Macaque Eyes
9
Western Blot Protein Analysis
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Total cell lysate was prepared in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM sodium fluoride, 2 mM EDTA, 0.1% SDS and a mixture of protease inhibitors). Western blot analysis was performed using the same amount of protein. Protein concentration was determined by the Bradford (Sigma-Aldrich, St. Louis, MO, USA) method and equal quantities were subjected to Western blot analysis. SDS-PAGE-separated proteins were electroblotted onto a nitrocellulose membrane. The blots were incubated overnight at 4 °C with the following primary antibodies: anti-ERRα (ab76228; 1:1000); anti-SR-BI (ab52629; 1:1000); anti-HMGCR (ab215365; 1:500); from Abcam, Cambridge, UK; anti-Cyclin D1 (sc-8396; 1:500), anti-PARP-1 (sc-7150; 1:2000) and anti-GAPDH (sc-32233; 1:10,000) from Santa Cruz Biotechnology, Dallas, TX, USA. Anti βactin (ab8226; 1:1000; Abcam) was used as a loading control. All antibodies were incubated with appropriate horseradish peroxidase conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were detected by the ECL Western blotting detection system (Santa Cruz Biotechnology, sc-2048). All the whole western blot figures can be found in the Supplementary Materials .
10
Western Blot Protein Analysis Protocol
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Total protein was isolated using RIPA lysis buffer (JRDUN, Shanghai, China). 25 μg protein of each sample was fractionated and transferred onto PVDF nitrocellulose membrane (HATF00010, Millipore, USA) for 12 h. The transfer method was semidry at 25 V on 100 mA current, lasted for 30 minutes, and the pore size of the PVDF membrane was 0.22 μm. Then, the membranes were probed with the primary antibodies overnight at 4°C, followed by the appropriate HRP-conjugated goat anti-rabbit IgG (A0208, Beyotime, China). Protein signals were analyzed using a chemiluminescence system. The antibodies details were provided as follows: TRIM37 (Ab95997, Abcam, UK, 1 : 1000), TRAF2 (Ab60169, Abcam, UK, 1 : 1500), SR-B1(Ab52629, Abcam, UK, 1 : 1000), ABCA1 (Ab18180, Abcam, UK, 1 : 1000), ABCG1 (Ab52617, Abcam, UK, 1 : 2000), NF-KB (#8242, CST, USA, 1 : 1000), H3 (#4499, CST, USA, 1 : 1000), and β-actin (#4970, CST, USA, 1 : 1000).
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