Tissue sections (5 μm, Leica RM2165) were placed in buffer at 70 °C for 3 h, diaphanized in xylol, re-hydrated and rinsed with distilled water, then heated at 90 °C immersed in a buffer citrate solution in a micro-oven. Endogenous peroxidase activity was blocked by hydrogen peroxide (H2O2, 3%) and blocked with normal horse serum (Vector Labs, Burlingame, CA, USA). Sections were incubated with primary antisera raised against P450arom anti-rabbit/mouse (1/400- ab18995, Abcam, Cambridge, MA, USA), NADPH-cytochrome P450 oxido-reductase (CPR) anti-mouse, rat, sheep, rabbit, guinea pig, hamster, cow, dog, human, pig, monkey (1/200- ab13513, Abcam, Cambridge, MA, USA), and P450c17 (1/200- Dr. Alan J. Conley, UC, Davis, California, USA), for 16 h. The primary antiserum was omitted for negative controls. Sections were rinsed, incubated with secondary anti-mouse/rabbit antisera ready-to-use (Immpress Universal Kit™,Vector Labs, Burlingame CA USA) followed by amplifier solution and developed with DAB (ImmPACT™ DAB, Vector Labs, Burlingame, CA USA). Sections were counter-stained with hematoxylin and mounted with coverslips. Images were captured with camera Olympus UTVO.5XC mounted on an Olympus BX61VS light microscope.
Ab18995
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab18995 is a polyclonal antibody that targets the Histone H3 (acetyl K9) protein. It is suitable for use in various research applications, such as Western blotting, immunohistochemistry, and chromatin immunoprecipitation (ChIP).
Automatically generated - may contain errors
Lab products found in correlation
Sc 542, by Santa Cruz Biotechnology (4 mentions)
Sc 8974, by Santa Cruz Biotechnology (3 mentions)
Ab13513, by Abcam (2 mentions)
3 3 diaminobenzidine (dab), by Fujifilm (2 mentions)
Sc 816, by Santa Cruz Biotechnology (2 mentions)
Fish sp15 500, by Thermo Fisher Scientific (2 mentions)
N histofine simple stain mouse max po, by Nichirei Biosciences (2 mentions)
Normal horse serum, by Vector Laboratories (1 mentions)
U tvo 5xc, by Olympus (1 mentions)
Bx61vs light microscope, by Olympus (1 mentions)
Rm2165, by Leica camera (1 mentions)
Immpact dab, by Vector Laboratories (1 mentions)
Ab6160, by Abcam (1 mentions)
Ab9486, by Abcam (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Sc 515120, by Santa Cruz Biotechnology (1 mentions)
Cm1850, by Leica camera (1 mentions)
G1213, by Wuhan Servicebio Technology (1 mentions)
Ab3259, by Abcam (1 mentions)
Ab190103, by Abcam (1 mentions)
18 protocols using ab18995
1
Immunohistochemical Localization of Steroidogenic Enzymes
2
Quantification of CYP19A1 and ESR1 in Testes
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Western blotting for CYP19A1 and ESR1 was performed as reported previously (45 (link)) on 80-d-old control and LCARKO testes. Blots were probed with primary antibodies ESR1 (sc-542; Santa Cruz Biotechnology, Dallas, TX, USA) and tyrosinated TUBA (α-tubulin) isoforms (ab6160; Abcam, Cambridge, MA, USA) or CYP19A1 [aromatase (Arom), ab18995; Abcam] and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ab9486; Abcam). Primary antibodies were detected using either donkey anti-rabbit IRDye 680RD and goat anti-rat IRDye 800CW or donkey anti-goat IRDye 680RD and donkey anti-rabbit IRDye 800CW (all LI-COR Biosciences, Lincoln, NE, USA). Detection was carried out using the LI-COR Odyssey imaging system (LI-COR Biosciences) according to the manufacturer’s instructions.
3
Ovarian Follicle Developmental Analysis
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The ovaries were quickly collected, cleaned of fat, fixed in 4% paraformaldehyde, dehydrated in 70% ethanol and embedded in paraffin. Then, the ovaries were serially sectioned into 5 μm thick sections (LEICA CM1850, Germany) and stained with hematoxylin and eosin (Beisuo Biotech Company, China). Follicles were counted in every fifth section and categorized into different developmental stages based on standards of classification.35 (link) Immunohistochemistry was performed as described previously.36 (link) The primary antibodies included anti-CYP19A1 (ab18995; Abcam) and anti-3β-HSD (sc-515120; Santa Cruz).
4
IHC Analysis of METTL3, CYP19A1, and ERβ
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Formalin-fixed, paraffin-embedded tissue was cut into 4 μm sections and analyzed by IHC, as described previously [14 (link)]. Briefly, tissue sections were incubated with anti-METTL3 (#ab195352, dil. 1:500, Abcam), anti-Aromatase (#ab18995, dil. 1: 500, Abcam), anti-ESR2 (#14007-1-AP, dil. 1:200, Proteintech) as primary antibodies after heat-induced epitope retrieval, followed by secondary antibody (diluted 1:100, G1213, Servicebio) incubation and diaminobenzidine. Slides were counterstained with hematoxylin. Then the researchers scored the expression levels of METTL3, CYP19A1, and ERβ in a double-blind manner, and the scoring system was rationalized according to the previous description [15 (link)].
5
Granulosa Cell Protein Expression Analysis
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Granulosa cells were seeded in six-well plates and transfected with overexpression plasmid, siRNA, or miRNA mimics for 48 h. Cells were harvested, washed with 1 × phosphate buffered saline (PBS), and lysed in RIPA lysis buffer. Equal amount of proteins were separated at sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After incubation with the indicated primary and secondary antibodies, signals were visualized by ECL. Membrane was then ready for scanning by Image studio system. Protein quantification was conducted by ImageJ software. The primary antibodies used were anti-CYP11A1 (1:1000; catalogue no. ab67355, Abcam, Cambridge, UK), anti-CYP19A1 (1:1000; catalogue no. ab18995, Abcam, Cambridge, UK), anti-SCAP (1:1000; catalogue no. ab190103, Abcam, Cambridge, UK), anti-SREBP1 (1:1000; catalogue no. ab3259, Abcam, Cambridge, UK), anti-Argonaute-2 (1:1000; catalogue no. ab186733, Abcam, Cambridge, UK), and anti-β-actin (1:10,000; catalogue no. 66009-1-Ig, Proteintech, Chicago, IL, USA). The goat anti-rabbit IgG (H + L)-HRP (1:5000; catalogue no. ab6721, Abcam, Cambridge, UK) were used as a secondary antibody.
6
Quantifying Adrenal Aromatase Levels
7
Immunohistochemical Analysis of Hormone Receptors
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Lesions were fixed in 4% paraformaldehyde and embedded in paraffin. Five‐micrometre tissue sections were mounted on slides followed by 15 minutes of boiling in sodium citrate (pH 6) for antigen retrieval and blocked with 10% goat serum (Vector Laboratories, Burlingame, CA, USA). Slides were incubated at 4°C overnight with anti‐ER‐α (1:300; sc‐542; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti‐ER‐ß (1:500; sc‐8974; Santa Cruz Biotechnology, Inc.), anti‐aromatase (1:700; ab‐18995; Abcam Inc., USA) and anti‐KRAS (1:400; ab‐216890; Abcam Inc., Cambridge, MA, USA) primary antibodies to determine protein expression, respectively. Slides were incubated 60 minutes at room temperature with biotinylated goat anti‐rabbit IgG (1:200; Vector Laboratories), and for detection, ABC Vectorstain Elite reagents with DAB plus H2O2 (Vector Laboratories) were used. Tissue sections were counterstained with haematoxylin (Sigma‐Aldrich, St. Louis, MO, USA). Images of stained sections were captured using Nikon Eclipse 80i microscope (Nikon).
8
Immunohistochemical Analysis of Steroid Receptors
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The serial sections of brain tissues were incubated with 10% normal goat serum to reduce background staining caused by the second antibody. The sections were then incubated with primary antibodies (1:1000) raised against rabbit polyclonal anti-AR (sc-816, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-ERα (sc-542, Santa Cruz Biotechnology), rabbit polyclonal anti-ERβ (sc-8974, Santa Cruz Biotechnology) and rabbit polyclonal anti-aromatase (ab18995, Abcam, Cambridge, MA) for 12 h under 4°C. The sections were then incubated with a secondary antibody, goat anti-rabbit lgG conjugated with biotin and peroxidase with avidin, using rabbit ExtrAvidin™ Peroxidase staining kit (Sigma Chemical Co., St. Louis, MO, USA) was performed, followed by visualizing with 30 mg 3,3-diaminobenzidine (Wako, Tokyo, Japan) solution in 150 ml of 0.05 Mol Tris-HCl buffer, pH 7.6, plus 30 µ l H2O2. The specificity of AR, ERα, ERβ and P450arom antibodies has been described in our previous studies.25 (link),30 The control sections were treated with normal rabbit serum instead of the primary antisera.
9
Immunohistochemical Analysis of Adrenal Gland
10
Immunohistochemical Detection of Aromatase
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Fixed tissues were deparaffinized and antigen retrieval was performed using a citrate-based buffer (pH 6.0 with detergent, Antigen Retrieval Buffer, PMB1-125, SpringBio), and endogenous peroxidases were quenched with 0.3% H2O2 in methanol for 15 min. After rinsing in phosphate buffered saline (PBS), slides were blocked with ProteinBLock (DPB-125, SpringBio) for 20 min at room temperature. The sections were then incubated in humidified chambers overnight at 4-8°C with the following primary antisera: anti-P450arom (1:50, rabbit polyclonal, ab18995, Abcam). Antisera were diluted in PBS prior to immunolabeling. After incubation with the primary antisera, slides were rinsed for 5 min in PBS and incubated with N-Histofine Simple Stain Mouse MAX PO (Nichirei Biosciences) for 30 min prior to detection using the DAB kit (DAB-125, SpringBio). For negative control, the primray antisera were replaced by PBS, and, for positive control, we followed other aromatase studies (Conley et al., 1996 (link)). Slides were then counterstained with hematoxylin and mounted in permount (FISH-SP15-500, Thermo Fisher Scientific).
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