Immunohistochemistry and immunofluorescence analysis were performed as described previously (Chen et al., 2021 ). After rehydration and antigen retrieval, the 5-μm sections were blocked with 5% BSA, incubated with the primary antibody for 1 h and the corresponding secondary antibody for 1 h. The following primary antibodies were used: MVH (Abcam, ab13840), SOX9 (Millipore, AB5535; Sigma, AMAB90795), Nucleoprotein (Sino biological, 40143-R001), CD3 (Abcam, ab11089), 3β-HSD (Santa Cruz, sc-30820). The secondary antibodies were: FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, 711-095-152), TRITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 715-025-151), TRITC-conjugated donkey anti-goat IgG (Jackson ImmunoResearch, 705-025-147). For immunohistochemistry, staining was visualized using a diaminobenzidine substrate kit, examined with a Nikon microscope, and images were captured by a Nikon DS-Ri1 CCD camera. For immunofluorescence, the sections were examined with a confocal laser scanning microscope (Carl Zeiss Inc, Thornwood, NY).
3β hsd
Manufactured by Santa Cruz Biotechnology
Sourced in United States
3β-HSD is a laboratory equipment product that plays a crucial role in the detection and measurement of 3β-hydroxysteroid dehydrogenase, an enzyme involved in steroid hormone biosynthesis. This product provides researchers with a reliable tool for quantifying the activity of this important enzyme.
Automatically generated - may contain errors
Lab products found in correlation
17β hsd, by Santa Cruz Biotechnology (3 mentions)
Confocal laser scanning microscope, by Zeiss (2 mentions)
P450scc, by Santa Cruz Biotechnology (2 mentions)
β actin, by Abcam (2 mentions)
Ds ri1 ccd camera, by Nikon (2 mentions)
Nucleoprotein, by Sino Biological (1 mentions)
Fitc conjugated donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Tritc conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit, by Merck Group (1 mentions)
Tissue total protein extraction kit, by Sangon (1 mentions)
Goat anti rabbit, by Santa Cruz Biotechnology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cyp11a1, by Cell Signaling Technology (1 mentions)
Lhcgr, by Santa Cruz Biotechnology (1 mentions)
Cyp17a1, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Bromodeoxyuridine (brdu), by Abcam (1 mentions)
Foxa2, by Abcam (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Histostain sp kit, by Zhongshan Golden Bridge Biotechnology (1 mentions)
12 protocols using 3β hsd
1
Immunohistochemical and Immunofluorescence Analysis
2
Protein Expression Profiling in Rat Reproductive Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from the uterus and ovaries of adult F1 female rats with a Tissue Total Protein Extraction Kit (Sangon Biotech, Shanghai, China). Western blot was performed as described by Farah et al. [36 (link)]. The primary antibodies used in the present study were: Esr1 (Abcam ab32063/1:1000, Cambridge, MA, USA), 3β-HSD (Santa Cruz sc-30820/1:500, Santa Cruz, CA, USA), and β-actin (Cell Signaling Technology #4967/1:2000, Boston, MA, USA). Secondary horse-radish peroxidase labelled antibodies were goat anti-rabbit (Sigma-Aldrich, A9169/1:10,000) or goat anti-rabbit (Santa Cruz sc-2004/1:6000). The bands were detected by a chemiluminescence WesternBright™ ECL Substrate kit (Advansta, Menlo Park, CA, USA). Bands were visualised and quantified by Tanon-5200 Chemiluminescent Imaging Analysis System (Tanon Science & Technology, Shanghai, China).
3
Testicular Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from testicular tissues with protein lysis buffer (RIPA: PMSF = 99:1), and determined the concentration by the BCA assay. After denaturation, protein samples were subjected to SDS-PAGE, electrophoretically transferred to membranes. The membrane was blocked in 5% skimmed milk in TBST buffer for 1 h, and then incubated with primary antibody overnight at 4 °C. StAR (1:1000) and CYP11A1 (1:1000) is from Cell Signaling Technology, 3β-HSD (1:500) is from Santa Cruz; LHCGR (1:500), CYP17A1 (1:1000) and GAPDH (1:5000) are from Proteintech. The membrane was incubated with the appropriate secondary antibodies for 1 h. Finally, we visualized and quantified the bands.
4
Immunohistochemical and Immunofluorescence Analysis of Uterine Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tissue specimens were fixed overnight in 10% neutral buffered formalin and then dehydrated in increasing concentrations of ethyl alcohol, followed by clearing of alcohol by xylene. Uterine slices were deparaffinized and incubated in citrate buffer for antigen retrieval by hyperbaric heating and then incubated overnight at 4 °C with the primary antibodies including Menin (Bethyl, 1:1000), COX2 (Santa Cruz, 1:200), Dtprp (homemade, 1:200), Ki67 (Servicebio,1:200), PL1 (Santa Cruz, 1:200), 3β-HSD (Santa Cruz, 1:200), p450scc (Santa Cruz, 1:200), PR (Cell Signaling Technology, 1:200), HAND2 (Santa Cruz, 1:200), p27 (Abcam,1:100), pH3 (Cell Signaling Technology, 1:200), ERK1/2 (Cell Signaling Technology, 1:200) and p-ERK1/2 (Cell Signaling Technology, 1:200). A Histostain-SP Kit (Zhongshan Golden Bridge Biotechnology) was applied to visualize the antigen. Immunofluorescence staining for OCT4 (Cell Signaling Technology, 1:200), β-Catenin (Abcam, 1:200), H3K27me3 (Cell Signaling Technology, 1:200), BrdU (Abcam, 1:500), PCNA (Santa Cruz, 1:200) and FOXA2 (Abcam, 1:500) was performed in paraffin-fixed sections and secondary antibody CyTM3 AffiniPure Goat Anti-Rabbit IgG(H + L) (Jackson ImmunoResearch,1:200) were used. The images were captured by Leica DM2500 light microscope. Antibodies with detailed information are listed in Supplementary Table 1 .
5
Rat Annexin A5 Purification and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The rat annexin A5 was purchased from GenScript (Piscataway, NJ, USA). Dulbecco's modified Eagle's medium (DMEM)/Ham's nutrient mixture F12 (DMEM/F12) was purchased from Invitrogen (Grand Island, NY, USA). Percoll, HEPES, collagenase type I were from Sigma Chemical Co. (St. Louis, MO, USA). Hanks’ balanced salt solution (HBSS) without Ca2+ and Mg2+, and penicillin-streptomycin were purchased from Life Technologies, Inc. (Paisley, Scotland, UK). PD-98059 and Reverse-Transcription System were purchased from Promega (Madison, WI, USA). Antibodies against StAR, P450scc, 3β-HSD, and 17β-HSD were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). β-actin antibody, horseradish peroxidase-conjugated goat anti-mouse, and goat anti-rabbit secondary antibodies were purchased from Boster (Wuhan, China).
6
Immunofluorescent Analysis of Leydig Cell Autophagy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The collected mouse testes were fixed with the animal testis tissue fixative (Servicebio, Wuhan, China), dehydrated, embedded in paraffin, and cut into 4-μm sections. When TM3 mouse Leydig cells reached confluence, they were exposed to 4% paraformaldehyde (Beyotime, Shanghai, China) for fixing at room temperature (20°C–25°C) for 20 min and exposed to 0.25% Triton X-100 in phosphate-buffered saline (PBS; Gibco) for permeabilizing at room temperature for 15 min. The sections and cells were then incubated with diluted antibodies against microtubule-associated protein 1A/1B-light chain 3 (LC3; Abcam, Cambridge, UK) and 3β-hydroxysteroid dehydrogenase (3β-HSD; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. Subsequently, the appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Bioss, Beijing, China) were added and incubated at room temperature for 90 min. Nuclear counterstaining was carried out by staining the sections and cells with DAPI after washing them with PBS. Images were captured at ×200 using an inverted microscope (Olympus). The mean fluorescence intensities of LC3 and 3β-HSD were determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
7
Testicular Cells Isolation and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh testes were collected and placed in 4℃PBS containing 10% BSA. Followed by removing the tunica albuginea, testis was squeezed gently through a 200 meshes gauze to acquire the single cell suspension. For ROS detection, cells were incubated with 5mM diacetyldichlorofluorescein (DCFDA) (Sigma, Japan) at 37℃ for 30min. Cells were then centrifuged at 2000r/min for 5min and re-suspended in PBS. This was repeated 3 times to remove the DCFDA. Finally, samples were analyzed by FACScalibur flow cytometer (Becton Dickinson, Heidelberg, Germany).
For 3βHSD positive cells detection, testicular cells were fixed in 2% formaldehyde for 15min., Cells were then mixed with 0.2% Triton X-100 for 30min to penetrate membranes. Then, primary antibody 3βHSD (Santa Cruz Biotechnology, USA) was added in cells at RT for 2 hours. Followed by rinsing the cells, the secondary antibody Alexa 594-conjugated donkey-anti goat antibodies (Invitrogen, USA) were added into cells at RT for 1 hour in dark. Then, cells were rinsed by PBS and analyzed by FACS calibur flow cytometer (Becton Dickinson, Heidelberg, Germany).
For 3βHSD positive cells detection, testicular cells were fixed in 2% formaldehyde for 15min., Cells were then mixed with 0.2% Triton X-100 for 30min to penetrate membranes. Then, primary antibody 3βHSD (Santa Cruz Biotechnology, USA) was added in cells at RT for 2 hours. Followed by rinsing the cells, the secondary antibody Alexa 594-conjugated donkey-anti goat antibodies (Invitrogen, USA) were added into cells at RT for 1 hour in dark. Then, cells were rinsed by PBS and analyzed by FACS calibur flow cytometer (Becton Dickinson, Heidelberg, Germany).
9
Immunohistochemical Analysis of Bovine Tissue Sections
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed tissues were embedded in paraffin according to routine laboratory protocols, cut into 4-μm-thick sections, and stained with hematoxylin and eosin. For immunohistochemistry and immunofluorescence, tissue sections were deparaffinized and rehydrated using routine procedures. Sections were incubated overnight at 4°C using primary antibodies specific against the following markers: BTV NS2 (38 (link), 39 (link)), vimentin (Dako Agilent), von Willebrand factor (Dako Agilent), CD3 (Dako Agilent), MX-1 (76 (link), 77 (link)), 3β-HSD (Santa Cruz Biotechnology), P450 Aromatase (AcrisAntibodies GmbH), inhibin α (Ventana Medical Systems), smooth muscle actin (Dako Agilent), and KI-67 (Dako Agilent). All of the antibodies were diluted in Antibody Diluents OP Quanto (Thermo Scientific) before use. Immunohistochemistry was carried out using Dako EnVision kit (Dako), and slides were counterstained with Mayer's hematoxylin. For immunofluorescence, secondary antibodies conjugated with fluorescent dyes were used (Alexa Fluor; Thermo Fisher Scientific). BTV NS2 was detected using the a tyramide signal amplification kit (Thermo Fisher Scientific) according to the manufacturer's protocol and as already described (37 (link)). Images were captured using Zeiss LSM710 confocal microscope and then analyzed by using Zen 2011 software (Zeiss).
10
Comprehensive Antibody Panel for Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies in this study: p62 (ab56416), KI67 (ab15580), BrdU (ab6326), DDX4 (ab13840), WT1 (ab89901), Lamp2 (ab65231), CYP17a1 (ab125022), Ubiquitin (linkage-specific K48) (ab140601), Ubiquitin (linkage-specific K63) (ab179434), were all purchased from Abcam (Cambridge, UK). Antibodies in this study: LC3B (2775), Caspase-3 (9662), Lamp1 (99,437), CYP11a1 (14,217), Star (8449), Ubiquitin (58,395), were all purchased from Cell Signaling Technology (Boston, USA). Antibodies in this study: Active caspase-3 (AC033), CYP19a1 (AF6231), PCNA (AF1363), were all purchased from Beyotime Biotechnology (Shanghai, China). Other antibodies including Foxl2 (Novus Biologicals, NB100-1277), β-actin (ABclonal, AC026), FSHR (Proteintech, 22665-1-AP), 3β-HSD (Santa Cruz Biotechnology, SC-30,820), USP5 (Proteintech, 10473-1-AP), HA (Abmart, M20021). Goat anti-rabbit FITC (ZF-0311), goat anti-mouse FITC (ZF-0312), and goat anti-mouse TRITC (ZF-0313)-conjugated secondary antibodies were purchased from Zhong Shan Jin Qiao (Beijing, China).
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!