Immunohistochemical staining of CD34 (ab81289, Abcam, UK), FSHR (orb213952, Biorbyt, UK), AMH (orb523061, Biorbyt, UK), PCNA (ab29, Abcam, UK), and caspase-3 (ab184787, Abcam, UK) was performed in D15 ovarian tissue. After fixation, the ovarian tissue was embedded into 4 nm thick sections, followed by dewaxing, rehydration, antigen retrieval, blocking, addition of the primary antibodies against CD34, FSHR, AMH, PCNA, and caspase-3, and counterstaining with the secondary antibody and hematoxylin. Finally, the images were collected under a microscopy system (Hamamatsu Photonics, Japan). CD34 staining was located in the ovarian stroma and corpus luteum. FSHR, AMH, PCNA, and caspase-3 were mainly located in the ovarian granulosa cells. Four fields of view were randomly selected for observation at 400x. CD34 was scored using MVD [29 (link)]. FSHR, AMH, PCNA, and caspase-3 were scored using the H score. According to the H score equation, H‐score∑Pi (i + 1), where i is the intensity staining (0 = negative, 1 = weak, 2 = medium, and 3 = strong). Pi is at each intensity (0–100%) percentage of stained cells. The H scores were measured in granulosa cells. The sections were independently scored by two pathologists. If there is a difference in the scores, reevaluate the slides and the two observers reached a consensus [30 (link)].
Ab184787
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab184787 is a lab equipment product offered by Abcam. It serves as a core laboratory tool, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (28 mentions)
Ab182858, by Abcam (22 mentions)
Pvdf membrane, by Merck Group (11 mentions)
Ripa lysis buffer, by Beyotime (9 mentions)
Ab205718, by Abcam (9 mentions)
Ab214430, by Abcam (9 mentions)
Ab8226, by Abcam (8 mentions)
Ab9485, by Abcam (8 mentions)
Ab194583, by Abcam (7 mentions)
Bca protein assay kit, by Beyotime (6 mentions)
Ab181602, by Abcam (6 mentions)
Ab182733, by Abcam (6 mentions)
Ab6721, by Abcam (6 mentions)
Ab196495, by Abcam (5 mentions)
Ab15580, by Abcam (5 mentions)
Ab8245, by Abcam (5 mentions)
Ab8227, by Abcam (5 mentions)
Ab16502, by Abcam (4 mentions)
Ab32124, by Abcam (4 mentions)
Ab202068, by Abcam (4 mentions)
102 protocols using ab184787
1
Immunohistochemical Analysis of Ovarian Markers
2
Immunohistochemical Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hippocampal tissue was paraffin-embedded, cut into 5 μm, and dehydrated by gradient ethanol. Then 3% H2O2 was used for incubation at room temperature for 30 min, and endogenous peroxidase was inactivated. 10% sheep serum was added for incubation at room temperature for 1 h, Next primary antibody anti-Bax (50599-2, Proteintech), anti-Bcl-2 (26593-1, Proteintech), and anti-caspase-3 (Ab184787, Abcam) were, respectively, added for incubation for 1 h, and secondary antibody was added for incubation for 1 h. After PBS rinsing, each tissue was added with 50 μl DAB chromogenic, re-dyed with hematoxylin, dehydrated, xylene permeated, and sealed. The positive expression of the protein was observed under a 100-fold and 400-fold microscope.
3
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from cells and uterine tissue with a protein extraction kit (KeyGEN, Changchun, China) according to protocols from the supplier. Protein concentration was determined using a bicinchoninic acid assay (KeyGEN). Equal concentrations of total protein were separated by SDS-PAGE (12%). Proteins were transferred onto polyvinylidene difluoride membranes and were blocked for two hours with TBST (50 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, and 0.1% Tween 20) containing 5% BSA. The membranes were incubated with primary antibodies diluted in TBST overnight at 4 °C. Antibodies against cytochrome C (ab133504), caspase-3 (ab184787), BAX, (ab32503), and Bcl-2 (ab182858) were from Abcam (Shanghai, China). After washing three times in TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for two hours at room temperature and then washed three times for 10 min. Protein bands were visualized by exposure to an enhanced chemiluminescence detection system imager (Tanon Biotech, Shanghai, China) with an enhanced chemiluminescence solution (DiNING, Beijing, China). The relative intensity of each band was assessed by Image J 1.47 v software.
4
Western Blot Analysis of Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse lung tissue and MLE-12 cells were lysed by RIPA lysate. Mouse lung tissue needs to be additionally put into a homogenizer to fully lyse. The protein lysate was then centrifuged (12,000 g, 15 min, 4°C) and the supernatant was collected. After being mixed into the loading buffer, the protein lysate was heated to 100°C for 5 min. Then, we made a 10% sodium dodecyl sulfate polyacrylamide gel and added 10 μL of protein lysate. After electrophoresis and membrane transfer, the protein is transferred to the polyvinylidene fluoride membrane. We used 5% bovine serum albumin-PBS-Tween to block nonspecific antigens for 1 h at room temperature. The membrane was then incubated with primary antibody dilutions (β-actin, ab8226, Abcam, Cambridge, MA, USA; IL-1β, ab254360, Abcam, Cambridge, MA, USA; tumor necrosis factor (TNF), ab1793, Abcam, Cambridge, MA, USA; SOD1, ab51254, Abcam, Cambridge, MA, USA; SOD2, ab68155, Abcam, Cambridge, MA, USA; caspase 3, ab184787, Abcam, Cambridge, MA, USA; p65, ab16502, Abcam, Cambridge, MA, USA; p-p65, ab86299, Abcam, Cambridge, MA, USA; IκBα, ab183503, Abcam, Cambridge, MA, USA;) at 4°C overnight. After washing the membrane, we incubated the membrane at room temperature for 1 h with the secondary antibody dilution (Abcam, Cambridge, MA, USA). Finally, we used chemiluminescent liquid for color development.
5
Protein Expression Analysis in Ovarian Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The homogenized ovarian tissues were lysed in RIPA lysis buffer containing a protease inhibitor. The supernatants obtained after centrifugation for 15 min at 13,000 rpm were then collected for western blotting. The protein concentration was detected by the Bradford test. Thereafter, the proteins were separated by Mini-PROTEAN® TGX™ (Bio-Rad, Hercules, CA, USA) and transferred onto PVDF membranes using a Trans-Blot Turbo Transfer System. The PVDF membranes were blocked with 5% horse serum at room temperature for 3 h and incubated at 4 °C overnight with rabbit polyclonal antibodies against caspase-3 (diluted 1:2000; ab184787, Abcam, Cambridge, UK), total PI3K (1:1000, ab191606, Abcam, Cambridge, UK), total AKT (1:500, ab8805, Abcam, Cambridge, UK), and total mTOR (1:800, Dg-Peptide Co., Hangzhou, China). Subsequently, the primary antibodies were incubated with the corresponding secondary antibodies (1:5000 sc-2357 and sc-516102 Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C. The bands of the target proteins were imaged using a Bio-Rad Gel Documentation System and analyzed using Image Lab software (Bio-Rad, USA).
6
Immunofluorescent Analysis of Spinal Cord Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The spinal cord tissues of each group were fixed with 4% paraformaldehyde at 4 °C. The spinal cord tissue was placed at the bottom of 50 mL centrifuge tube and dehydrated in 10%, 20%, and 30% sucrose solution. Twelve micrometers frozen sections were made and detected. All sections were blocked with blocking solution [10% goat serum, 3% bovine serum albumin (BSA), and 0.1% Triton X-100] for 1 h at 37 °C and then incubated overnight at 4 °C with Rabbit-anti-Active-Caspase3 (1:500, ab49822, Abcam), Rabbit-anti-Caspase3 (1:500, ab184787, Abcam), Rabbit-anti-LC3 (1:100, ab128025, Abcam), Rabbit-anti-LAMP2 (1:400, L0668, Sigma), Rabbit-anti-Bcl-2 (1:500, NB100-56101, NOVUS) and Rabbit-anti-Bax (1:500, ab12503, Abcam); 0.01 M phosphate buffer saline (PBS) was used to wash them for 10 min at 3 times and followed by incubating with a mixture of FITC- or Cy3-conjugated secondary antibodies for 2 h at room temperature and then being washed again with PBS for 10 min at 3 times. The stained sections were examined with a Leica fluorescence microscope (Leica DM 5000B, Germany).
7
Western Blot Analysis of Apoptosis and Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HUVECs were lyzed using RIPA lysis buffer (Beyotime, Shanghai, China) and cell supernatants were collected by centrifugation. The protein concentration was determined using BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples were isolated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA, membranes were incubated with primary antibodies against Bcl-2 (Abcam, ab32124, 1:1000), Bax (Abcam, ab243140, 1:500), Cleaved caspase-3 (Abcam, ab2302, 1:500), Caspase-3 (Abcam, ab184787, 1:2000), Beclin-1 (Abcam, ab207612, 1:2000), Atg5 (Abcam, ab108327, 1:10,000), p62 (Abcam, ab207305, 1:1000), GRP78 (Abcam, ab108615, 1:10,000), CHOP (Abcam, ab11419, 1:1000), Caspase-12 (Abcam, ab62484, 1:2000), XBP-1 (Abcam, ab220783, 1:1000) and GAPDH (Abcam, ab32124, 1:1000) overnight at 4°C. Membranes were washed in TBST, followed by incubation with secondary antibodies (Abcam, ab205718, 1:50,000; Abcam, ab205719, 1:20,000) for 1 h at room temperature. Protein bots were visualized using an enhanced chemiluminescence (ECL) detection kit (Beyotime, Shanghai, China).
8
Western Blot Analysis of Immune Cell Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD4+ T cells and CD19+ B cells from mice were lysed through the RIPA buffer (WB038, GEFAN, China) [18 (link)]. The extracted protein was centrifuged and then quantified with the help of the BCA kit (23225, Thermo Scientific, USA). After being electrophoresed, they were transferred to the PVDF membrane before being blocked with 5% skim milk. Next, we utilized primary antibodies to treat the membrane at 4℃ all night. After that, second antibody was used to treat the membrane at 37℃ for 90 min. After rinsing, the reactive bands were reacted with the color reagent (34075, PIERCE, USA) in a gel imaging system (610020-9Q, Clinx, China). The primary antibodies of RUNX1 (1:1,000, ab240639, 48 kDa), FOXP3 (1:1,000, ab20034, 50 kDa), Bax (1:8,000, ab32503, 21 kDa), Bcl-2 (1:2,000, ab196495, 26 kDa), cleaved caspase-3 (1:5,000, ab214430, 17 kDa), caspase-3 (1:2,000, ab184787, 32 kDa), and GAPDH (1:10,000, ab181602, 36 kDa), and the second antibodies of goat anti-rabbit IgG H&L (HRP) (1:2,000, ab205718) and goat anti-mouse IgG H&L (HRP) (1:2,000, ab205719) were gained from Abcam (UK). GAPDH was exploited to the housekeeping gene. The protein level was normalized to GAPDH, and presented relative to control cells (set as 1).
9
Quantifying Tumor Angiogenesis and Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mice (n = 6/group) were treated as described above and at day 17 post-implantation tumors were harvested, frozen and cut into tissues sections. Tissue sections were stained with CD31 antibody (abcam, ab281583)/Ki67 antibody (abcam, ab15580)/cleaved caspase -3 (abcam, ab184787) followed by secondary anti-rat IgG conjugated to HRP (GE Healthcare, Piscataway, NJ). CD31+/Ki67+/cleaved caspase-3+ staining was revealed with 3,3′-diaminobenzidine (DAB) histochemistry (Vector Laboratories, Burlingame, CA). Sections were counterstained with hematoxylin (Sigma, St. Louis, MO). Tumor microvessel density was quantified for all treatment groups. At least 6–10 representative 40X fields per view were captured as epifluorescent digital images using a Spot digital camera (Spot Diagnostic instruments, Sterling, MI). To calculate microvessel density, area occupied by CD31-positive microvessels and total tissue area, per section were quantified using ImageJ software (NIH, Bethesda, MD). Microvessel density was then calculated as a percentage of CD31 stained per tumor section.
10
Western Blot Analysis of Macrophage Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins were isolated from liver macrophages and cultured RAW264.7 cells by lysing them in RIPA buffer containing 1% PMSF (Beyotime). The protein concentration was determined using a BCA protein kit (Beyotime). Based on the protein concentration, equal amounts of protein were separated by SDS/PAGE and transferred onto a PVDF membrane (Millipore Corp.) followed by blocking. The following primary antibodies were used in this study: anti‐STING (19851‐1‐AP; Proteintech), anti‐BAX (ab32503; Abcam), anti‐BAK (#578; CST), anti‐Bcl2 (ab182858; Abcam), anti‐p65 (#8242S; CST), anti‐p‐p65 (#3033S: CST), anti‐TBK1 (ab40676; Abcam), anti‐p‐TBK1 (#5483S; CST), anti‐caspase3 (ab184787; Abcam), anti‐IRF3 (#11904S; CST), anti‐p‐IRF3 (#37829S; CST), and anti‐β‐actin (7D2C10; Proteintech). The protein bands were visuzed using an enhanced chemiluminescence detection system (Bio‐Rad) and quantified after normzation to internal control β‐actin using the ImageJ software (NIH).
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!