Brain tissue with the range of +2.76 mm to −3.24 mm/Bregma was stained from each brain, and 3 random nonoverlapping fields were assessed from each section by a blinded observer using Image-Pro Plus 6.0 software. For immunohistochemical analysis for p-Akt, p-mTOR of the brain tissue was performed on representative serial paraffin-embedded sections as previously described. They were stained with rabbit polyclonal phospho-Akt antibody (CST, USA, 1 : 100) or rabbit polyclonal phospho-mTOR antibody (CST, USA, use a concentration of 5 μg/ml) followed by anti-antibody of HRP-conjugated goat anti-rabbit IgG H&L (1 : 7500 in TBS-T, Abcam, UK). Standard fluorescent immunohistochemistry procedures and the primary and secondary antibodies indicated below were used to treat the brain tissue for immunofluorescence: rabbit polyclonal anti-LC3 antibodies (Abcam, UK, use a concentration of 1 μg/ml) and HRP-conjugated goat anti-rabbit IgG H&L (1 : 7500 in TBS-T, Abcam, USA). PC12 cells were fixed by formaldehyde and blocked by Donkey serum and then were processed with standard immunofluorescent techniques using primary and secondary antibodies of goat polyclonal to NLRP3 (Abcam, UK, use a concentration of 10 μg/ml), rabbit monoclonal to NF-κB p65 (Abcam, UK, 1 : 100), and secondary antibody. The nuclear area was defined according to DAPI staining.
Hrp conjugated goat anti rabbit igg h l
Manufactured by Abcam
Sourced in United States, United Kingdom
HRP-conjugated goat anti-rabbit IgG H&L is a secondary antibody that binds to rabbit immunoglobulin G (IgG) heavy and light chains. The antibody is conjugated to horseradish peroxidase (HRP), which can be used for signal detection in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Hrp conjugated goat anti mouse igg h l, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti nlrp3 antibody, by Abcam (2 mentions)
Anti caspase 1 antibody, by Abcam (2 mentions)
Anti gapdh antibody, by Abcam (2 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg h l, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
Gapdh, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab6528, by Abcam (2 mentions)
Ab134146, by Abcam (2 mentions)
Ab32358, by Abcam (2 mentions)
Ab51608, by Abcam (2 mentions)
Ab54365, by Abcam (2 mentions)
Ab76417, by Abcam (2 mentions)
Ab6258, by Abcam (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Ab92547, by Abcam (2 mentions)
27 protocols using hrp conjugated goat anti rabbit igg h l
1
Immunohistochemical Analysis of Brain Tissue
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and tissue homogenates were harvested and lysed in RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF). The protein concentration in the lysate was quantified using the BCA Kit. The cell lysates were loaded onto an SDS-PAGE gel for electrophoresis. After transfer of the proteins onto a piece of nitrocellulose membrane, targets were detected by western blotting using primary antibodies, including NEK6 antibody (1 : 2000, catalog number: 10378-1-AP, Proteintech, Wuhan, China), GAPDH antibody (1 : 10,000, catalog number: 10494-1-AP, Proteintech), secondary antibody, HRP-conjugated goat anti-mouse IgG (H + L) (1:10,000, catalog number: ab205719, Abcam, United States), HRP-conjugated goat anti-rabbit IgG (H + L) (1:10,000, catalog number: ab205718, Abcam), and an ECL Chemiluminescent Substrate Reagent Kit (Invitrogen, catalog number: WP20005, Shanghai, China). Protein expression levels were quantified using ImageJ software. The experiments were performed in triplicate.
3
DOCK1 Expression in AML Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the proteins were obtained from AML tissues and cells using the RIPA lysis buffer and were quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, USA). SDS-PAGE (8%) was used to separate the protein, and the concentration of loaded protein samples was 40 μg/lane. After performing electrophoresis at constant pressure, the separated protein bands were electro-transferred onto the PVDF membrane (Millipore, USA) with constant current. The membrane was then washed with PBST (0.1% Tween-20/PBS solution) three times and blocked with 5% BSA for 2 h at room temperature. Subsequently, the rabbit polyclonal anti-DOCK1 (Cat#: ab97325, Abcam, USA) and rabbit monoclonal anti-GAPDH (Cat#: EPR16891, Abcam, USA) were employed for primary antibody incubation overnight at 4 °C. HRP-conjugated Goat anti-rabbit IgG H&L (Cat#: ab181602, Abcam, USA) was used for secondary antibody incubation for 2 h at room temperature. After repeated washing with PBST, the blots were visualized using the enhanced chemiluminescence kit (Thermo Fisher Scientific, USA).
4
Ang II and Pitavastatin Modulate Endothelial Function
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ang II was purchased from APExBIO Technology LLC, whilst pitavastatin was purchased from HL Genomics, Co., Ltd. Antibodies were purchased as follows: Anti-phosphorylated (p)-endothelial NO synthase (eNOS; cat. no. ab215717; 1:1,000; Abcam), anti-eNOS (cat. no. ab76198; 1:1,000; Abcam), anti-endothelin (ET)1 (cat. no. ab2786; 1:1,000; Abcam), anti-Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), anti-Bax (cat. no. ab32503; 1:1,000; Abcam), anti-cleaved caspase-3 (cat. no. ab2302; 1:1,000; Abcam), anti-cleaved poly (ADP-ribose) polymerase 1 (PARP1; cat. no. ab32064; 1:1,000; Abcam), anti-ERK5 (cat. no. ab40809; 1:1,000; Abcam), anti-GAPDH (cat. no. ab181602; 1:2,000; Abcam), HRP-conjugated goat anti-mouse IgG H&L (cat. no. ab205719; 1:10,000; Abcam), HRP-conjugated goat anti-rabbit IgG H&L (cat. no. ab205718; 1:10,000; Abcam) and Alexa Fluor® 488 goat anti-rabbit IgG H&L (cat. no. ab150077; 1:10,000; Abcam).
The following kits were purchased as follows: NO Colorimetric assay kit (cat. no. E-BC-K035-S; Elabscience Biotechnology, Inc.), reactive oxygen species (ROS) colorimetric assay kit (cat. no. E-BC-K138-F; Elabscience Biotechnology, Inc.), TNF-α ELISA kit (cat. no. ADI-901-099; Enzo Life Sciences, Inc.), IL-1β ELISA kit (cat. no. EHC002b.48; NeoBioscience Technology Co., Ltd.) and the IL-6 ELISA kit (cat. no. ADI-901-033; Enzo Life Sciences, Inc.).
The following kits were purchased as follows: NO Colorimetric assay kit (cat. no. E-BC-K035-S; Elabscience Biotechnology, Inc.), reactive oxygen species (ROS) colorimetric assay kit (cat. no. E-BC-K138-F; Elabscience Biotechnology, Inc.), TNF-α ELISA kit (cat. no. ADI-901-099; Enzo Life Sciences, Inc.), IL-1β ELISA kit (cat. no. EHC002b.48; NeoBioscience Technology Co., Ltd.) and the IL-6 ELISA kit (cat. no. ADI-901-033; Enzo Life Sciences, Inc.).
5
Western Blot Analysis of USP32 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
U-251 MG and U-87 MG cells were lysed using RIPA buffer (Vazyme). The protein concentrations of samples were measured using a bicinchoninic acid (BCA) protein quantification kit (Abcam, Shanghai, China). Samples (12 μg/lane) were loaded into a 12% SDS-PAGE gel for electrophoresis and then transferred onto a PVDF membrane (Roche, Basel, Switzerland). The membrane was incubated with primary antibody Anti-USP32 (1:1000, CAT#ab251903, Abcam) or Anti-GAPDH (1:2000, CAT#ab9485, Abcam) at 4 °C overnight, followed by incubation with secondary antibody HRP-conjugated Goat Anti-Rabbit IgG H&L (1:2000, CAT#ab6721, Abcam) at 28 °C for 30 min. The signals were visualized using the ECL detection system (Thermo Fisher Scientific, Waltham, MA, USA) and quantified by densitometry using Image J v1.48u.
6
PHB Immunohistochemical Analysis in Kidney
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PHB levels were further examined using immunohistochemistry. The kidney tissues from control and experimental groups of 2, 6 and 8 h following brain death were fixed with 4% paraformaldehyde for 24 h, dehydrated and embedded in paraffin. Paraffin blocks were sectioned into 5 µm-thick slices and fixed with a chilled 1:1 mixture of methanol:acetone for 5 min following pretreatment with 0.3% hydrogen peroxide for 20 min at room temperature. For PHB-specific staining, the sections were incubated by primary polyclonal rabbit antibody (cat. no. ab75766; 1:1,000; Abcam) for 1 h at 37°C. Subsequently, incubation with HRP-conjugated goat anti-rabbit IgG H&L (1:10,000; Abcam, Cambridge, MA, USA) at 37°C for 30 min was followed by reaction with 3,3-diaminobenzidine substrate solution and counterstaining with hematoxylin. A total of five random fields of view of each stained section were pictured and analyzed using morphometric software (MIAS-2000 medical image analysis system; Beijing University of Aeronautics and Astronautics, Beijing, China) by an investigator blind to the study group.
7
Mammalian Cell Lysis and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mammalian cells cultured near confluence in T75 flasks were washed twice with cold PBS and scraped into 200 µl of Pierce 1x RIPA buffer (Thermo Scientific), supplemented with protease inhibitor cocktail (Millipore Sigma), and then transferred to 1.5 ml microtube kept on ice. Cells were vortexed briefly and disrupted by trituration 10× with a 23 G ¼ inch syringe needle (Becton, Dickinson and Co. Thermo Scientific). To remove unbroken cells and cellular debris, lysates were spun at 17,000 × g for 10 min and the supernatant was collected for downstream applications, as well as for determination of protein concentration by the BCA Protein Assay Kit (Thermo Scientific).
For running Western blots, 30 µg of cell lysates in Sample Buffer was loaded per each well of 4–20% Precast Protein Gel (BioRad, Hercules, CA) and then transferred to PVDF membrane. For blocking, 3% BSA (Sigma-Aldrich) in TBS buffer was used, while antibodies were diluted in 1% BSA in TBS-T. For detection of CD63 protein, we used either anti-mouse CD63 (ab217345) or anti-human CD63 (ab134045) (Abcam, Waltham, MA); for detection of NHE1, the Anti-Na+/H+ Exchanger 1 (extracellular) was used from Alomone Labs (ANX-010, Jerusalem, Isreal); for secondary antibodies, the HRP-conjugated goat anti-rabbit IgG H&L (ab6721, Abcam) was used; primary antibodies were used at 1:2000 and secondary antibodies at 1:10,000 dilutions.
For running Western blots, 30 µg of cell lysates in Sample Buffer was loaded per each well of 4–20% Precast Protein Gel (BioRad, Hercules, CA) and then transferred to PVDF membrane. For blocking, 3% BSA (Sigma-Aldrich) in TBS buffer was used, while antibodies were diluted in 1% BSA in TBS-T. For detection of CD63 protein, we used either anti-mouse CD63 (ab217345) or anti-human CD63 (ab134045) (Abcam, Waltham, MA); for detection of NHE1, the Anti-Na+/H+ Exchanger 1 (extracellular) was used from Alomone Labs (ANX-010, Jerusalem, Isreal); for secondary antibodies, the HRP-conjugated goat anti-rabbit IgG H&L (ab6721, Abcam) was used; primary antibodies were used at 1:2000 and secondary antibodies at 1:10,000 dilutions.
8
Spinal EZH2 Expression in Rats
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were anaesthetized with an intraperitoneal injection of 5% chloral hydrate (400 mg/kg). Spinal L4-6 tissues were dissected from rats on ice and stored at −80 °C until use. Spinal cDNAs were fully obtained through reverse transcription. The expression of the EZH2 mRNA relative to β-actin was calculated using the 2−ΔΔCt method.
For Western blot analysis, 50 µg of protein from the spinal homogenate was obtained, separated on an SDS–PAGE gel and transferred to a PVDF membrane. Membranes were blocked with 5% non-fat milk for at least 1 h and incubated with polyclonal rabbit anti-EZH2 (1:500, Abcam), anti-H3K27Tm (1:500, Abcam) or anti-β-actin (1:2000, Abcam) primary antibodies overnight at 4 °C; an HRP-conjugated goat antirabbit IgG H&L (1:5000, Abcam) secondary antibody was subsequently incubated with the membrane. The resulting bands were analysed using ImageJ software.
For Western blot analysis, 50 µg of protein from the spinal homogenate was obtained, separated on an SDS–PAGE gel and transferred to a PVDF membrane. Membranes were blocked with 5% non-fat milk for at least 1 h and incubated with polyclonal rabbit anti-EZH2 (1:500, Abcam), anti-H3K27Tm (1:500, Abcam) or anti-β-actin (1:2000, Abcam) primary antibodies overnight at 4 °C; an HRP-conjugated goat antirabbit IgG H&L (1:5000, Abcam) secondary antibody was subsequently incubated with the membrane. The resulting bands were analysed using ImageJ software.
9
Evaluating Neuroinflammation Protein Expressions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the indicated 24 h treatment with Aβ1‒42, BV2 cells were treated with various formulations (100 nmol/L Rapa, 500 nmol/L GP-17) for 24 h. The protein samples were harvested as previously aforementioned. Western Blotting analysis was then performed to evaluate the expression of neuroinflammation-associated proteins. The used antibodies were as followed: anti-NLRP3 antibody (Abcam, 1:1500), anti-Caspase1 antibody (Abcam, 1:1000), anti-GAPDH antibody (Abcam, 1:5000), and HRP-conjugated Goat Anti-Rabbit IgG H&L (Abcam, 1:5000).
10
Western Blot Analysis of LPS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five μl of each LPS was separated by electrophoresis as described above. LPS was transferred to 0.2 μm polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo blotting system (Bio-Rad) with a high-molecular-weight (MW) program (1.3A up to 25 V for 10 min). The membranes were blocked overnight in 5% BSA in PBS plus 0.1% Tween 20 (PBST), then incubated with 1 μg/ml KM467 or polyclonal anti-O25 typing sera (Abcam, diluted 1:10) in 0.5% BSA in PBST for 2 h at RT. The membranes were washed 3 × 10 min in PBST, then incubated with 0.1 μg/ml HRP-conjugated Goat Anti-Human IgG H&L or HRP-conjugated Goat Anti-Rabbit IgG H&L (Abcam) in 0.5% BSA in PBST for 1 h at RT. The membranes were washed 6 × 10 min in PBST, then developed with SuperSignal West Pico PLUS (Thermo Scientific).
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!