The AR expression in BC tissues were measured by immunohistochemistry (IHC). In brief, samples were fixed at 60°C for 1 hour and deparaffinized in xylene solutions, followed by rehydration. Antigen retrieval in slides was conducted by T Cell Conditioning 1 (CC1) and incubated with 3% hydrogen peroxide for 30 minutes. After blocking with 5% goat serum, samples were incubated with primary AR antibody (ab133273, 1:100, Abcam) at 4°C overnight, followed with secondary goat anti-rabbit IgG H&L (HRP) antibody (ab205718, 1: 5000, Abcam) at 37°C for 30 minutes. Finally, after washing by PBS for 3 times and TBST for once, the slides were stained with diaminobenizidine (DAB). Bound antibody on the array was determined by an OptiView DAB detection kit. Counterstain of the samples were conducted using hematoxylin for 1 minute, followed with bluing by PBS. The pathologic scoring of AR was semiquantitatively evaluated. The score for stained area was defined as follows: 0 for none, 1 for <1/100, 2 for 1/100 to 1/10, 3 for 1/10 to 1/3, 4 for 1/3 to 2/3 and 5 for more than 2/3. IHC score was finally graded as negative for 0–2, weak for 3–4, moderate for 5–6, and strong for 7–8.
Ab133273
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab133273 is a laboratory product offered by Abcam. It is a specific type of lab equipment, but no further details about its core function can be provided in an unbiased and factual manner without the risk of extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (3 mentions)
Ab45179, by Abcam (3 mentions)
Ab201687, by Abcam (3 mentions)
Fv1000 d microscope, by Olympus (3 mentions)
Ab8226, by Abcam (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ab75813, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab16667, by Abcam (1 mentions)
Ac007, by ABclonal (1 mentions)
Ab32042, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Ab32084, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Ab18197, by Abcam (1 mentions)
Lieca qwin 500 image analyzer, by Leica camera (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
25 protocols using ab133273
1
Immunohistochemical Evaluation of AR Expression
2
Protein Expression Analysis in Dermal Papilla Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were isolated from DPCs using a RIPA protein lysis buffer containing a protease inhibitor cocktail (Roche, Switzerland). Protein concentrations were determined using a BCA protein assay kit (Pierce, IL, United States), measuring the absorbance at 562 nm. Total protein (20 μg) was subjected to SDS-PAGE, and the separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore, MA, United States). The membrane was blocked in 5% bovine serum albumin (BSA) for 1 h and probed with appropriate primary antibodies overnight at 4°C. Primary antibodies against the following proteins were used: AR (1:2,000; ab133273, Abcam, Cambridge, United Kingdom), neural cell adhesion molecule (NCAM; 1:2,000, ab75813, Abcam), β-catenin (1:2,000; ab32572, Abcam), Ki67 (1:2,000; ab16667, Abcam), cleaved caspase-3 (1:2,000; ab32042, Abcam), β-actin (1:5,000; ab8226, Abcam), and alpha-tubulin (1:5,000; AC007, ABclonal, MA, United States). After washing with Tris Buffered saline Tween, the blots were incubated with the corresponding secondary antibody (1:1,000; ab150077, Abcam) for 1 h at 20–25°C and photographed using an Odyssey infrared fluorescent scanning imager (Bio-Rad, CA, United States).
3
Immunohistochemical Analysis of Tumor Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumors were resected in 2-μm thickness and fixed in 4% paraformaldehyde, embedded with paraffin. The cross-sectioned tissues were stained with H&E to observe histology. For immunohistological analysis, paraffin sections were dewaxed in xylene and rehydrated in graded ethanol, followed by incubation with non-specific protein blocking solution 1% bovine serum albumin (Thermo Fisher Scientific, # 11021037;, Waltham, USA) in PBS for 45 min at room temperature, and incubated with primary antibodies against AR (1:300, Abcam, ab133273, Cambridge, UK) or Paxillin (1:400, Abcam, ab32084) overnight at 4 °C. For negative controls, blocking solution was added instead of the primary antibody. Then the slides were incubated with EnVision-HRP secondary antibody for 1 h. a. The slides were developed with diaminobenzidine detection kit (Dako cytomation, Denmark). After being counterstained with haematoxylin, the samples were visualized under a light microscope (Olympus, Tokyo, Japan).
4
Immunostaining Procedure for Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunostaining was performed as described before [31 (link)]. Briefly, cells were grown on cover glasses and fixed for 15 min with 4% paraformaldehyde (PFA) solution at room temperature. Cells were washed twice with PBS and then permeabilized with 0.1% Triton-X100 in PBS for 10 min. Then, cells were incubated with a blocking buffer (PBS with 5% BSA). Primary antibody was applied at the specified dilution in blocking solution overnight at 4 °C. The following morning, cells were washed twice with PBS and incubated with the appropriate fluorescent secondary antibody (in blocking solution) for 30 min in the dark. Coverslips were then washed three times with PBS, mounted with DAPI, and examined under Olympus FV1000D microscope. The following antibodies were used for immunostaining: ARHGEF2 (ab201687, Abcam), CHGA (ab45179, Abcam), and AR (ab133273, Abcam).
5
Immunohistochemical Analysis of AR and PCNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immune-histochemical analysis of AR and PCNA were done according to Abdel-Rahman et al. (2017) . The tissue specimens were deparaffinized and rehydrated then pretreated with citrate buffer pH 6 for 20 min for antigen retrieval. The sections were incubated with primary antibodies of rabbit polyclonal anti-androgen receptor antibody (ab133273; Abcam, Cambridge, UK) diluted 1:500 and Rabbit polyoclonal anti-PCNA antibody (ab18197; Abcam, Cambridge, UK) diluted 1:4000 for three hours in a humidified chamber. The sections were incubated with secondary (HRP) antibody (ab205718; Abcam, Cambridge, UK). DAB (Sigma) was used as chromogen to visualized reaction. Finally, the slides were counterstained with Mayer hematoxylin and mounted with DPX. The images were analyzed by Lieca Qwin 500 Image Analyzer (Leica, Cambridge, England). In each group, five sections were examined. Color density of the immunopositive cells (dark brown) was evaluated.
6
Protein Quantification and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentration was assessed by bicinchoninic acid assay as above. Samples were denatured at 95°C for 5 minutes in Laemmli's sample buffer containing 2.5% 2‐mercaptoethanol. Equal protein amounts of denatured samples were loaded on 4% to 15% polyacrylamide gels. Proteins were separated by SDS‐PAGE and transferred to nitrocellulose membranes. After blocking with 5% w/v BSA (bovine serum albumin), the membrane was incubated with primary antibodies to p‐Smad2 (1:500) (Cell Signaling Technology), Smad2 (1:1000) (Cell Signaling Technology), Gapdh (1:1000) (Cell Signaling Technology), or androgen receptor (1:500) (ab133273, Abcam, Cambridge, UK) overnight. Protein bands were visualized with secondary IgG HRP conjugates (1:3000) (Cell Signaling Technology) and enhanced ECL (EMD Millipore Corporation, Burlington, MA). Band intensities were measured by densitometry scanning using ImageJ software (National Institute of Health, Bethesda, MD) and were normalized to Gapdh as a loading control. Because p‐SMAD signals were not detected in the vehicle control Fbn1 SMC, results were expressed as fold difference compared with TGF‐β treated Fbn1 SMC. Experiments included n=6 to 9 mice per group (WT males=6; WT females=6; Fbn1 males=6; Fbn1 females=6; flutamide‐treated Fbn1 males=9; vehicle control‐treated males=9).
7
Western Blot Analysis of Androgen Receptor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using RIPA Lysis Buffer (Thermo Scientific™, New York, USA). The full set of electrophoretic instruments was from Bio-Rad (USA). Anti-AR antibody (1:1000, ab133273, Abcam, Cambridge, UK), anti-β-tubulin antibody (1:5000, ab15568) and goat polyclonal secondary antibody to rabbit IgG (HRP-conjugated) (1:5000, ab6721) were used.
8
Immunostaining Procedure for Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunostaining was performed as described before [31 (link)]. Briefly, cells were grown on cover glasses and fixed for 15 min with 4% paraformaldehyde (PFA) solution at room temperature. Cells were washed twice with PBS and then permeabilized with 0.1% Triton-X100 in PBS for 10 min. Then, cells were incubated with a blocking buffer (PBS with 5% BSA). Primary antibody was applied at the specified dilution in blocking solution overnight at 4 °C. The following morning, cells were washed twice with PBS and incubated with the appropriate fluorescent secondary antibody (in blocking solution) for 30 min in the dark. Coverslips were then washed three times with PBS, mounted with DAPI, and examined under Olympus FV1000D microscope. The following antibodies were used for immunostaining: ARHGEF2 (ab201687, Abcam), CHGA (ab45179, Abcam), and AR (ab133273, Abcam).
9
Androgen Receptor Expression in Gubernaculum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
E19 gubernaculum pairs were dissociated overnight at 4°C in 5 mg/ml collagenase with 2.5 mM CaCl. Samples were triturated and then plated on collagen or poly-L lysine (PLL)/laminin-coated 6-well tissue culture plates. Cells were then incubated for a total of 6 days in DME/F12 containing 18% fetal calf serum (FCS), rat basic fibroblast growth factor (bFGF; 2.5 ng/ml), rat epidermal growth factor (EGF; 10 ng/ml), dexamethasone (0.4 ug/ml), and penicillin-streptomycin supplemented with 0, 0.1, 1.0, or 10.0 nM DHT. Media was changed on each of the 6 days of culture. Immunofluorescent staining of AR in gub cells was carried out using 1:200 dilution of the rabbit anti-AR antibody (Abcam cat# ab133273) in PBS at 4°C overnight. Cells were washed 3X with PBS and then incubated with donkey anti-rabbit Alexaflour 555 and Dapi (Fisher cat#EN62248) to visualize nuclei. On day 6, 5 random areas were captured from each treatment protocol using an Olympus BX-60 florescence microscope with an Evolution QEi 12-bit digital camera (Media Cybernectics) and a 20X objective. AR+ cells were counted manually using Image J software (National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/ ).
10
Western Blot Analysis of Prostate Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction from frozen prostate samples for Western blot analysis has previously been described [49 (link)]. Western blots were prepared using 15–20 µg of protein lysate, blocked with 5% BSA in 1 × TBS / 0.1% Tween-20 for 1 h and incubated at 4 °C overnight with primary antibodies against AR (1:1000, ab133273, Abcam), p16INK4A (1:2000, ab211542, Abcam), CDK4 (1:200, sc-260, Santa Cruz), p53 (1:1000, CST#2524, Cell Signaling), β-Actin (1:1000, CST#4967, Cell Signaling) and β-Tubulin (1:2000, CST#2146, Cell Signaling). Western blots were quantified using ImageJ2.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!