Skin samples were fixed in 10% formalin for immunohistochemistry. Fixated samples were rinsed in tap water, dehydrated with ethanol at increasing concentrations, cleaned with xylene, and embedded in paraffin. In descending grades of ethanol, paraffin sections were deparaffinized and rehydrated. Epitopes were extracted by boiling sections in citrate buffer (pH 6.0) in the microwave. Endogenous peroxidase was inhibited by soaking slides in ethanol containing 3% hydrogen peroxide. For 60 minutes at room temperature, sections were incubated with rabbit anti-human MIF (Chongqing Biospes Co., Ltd, Chongqing, China, dilution 1:50). After washing in TBS-T, sections were incubated for 2 hours at room temperature with HRP-conjugated goat anti-rabbit secondary antibodies (Thermofisher Scientific, Rockford, IL, USA) at a dilution of 1:250. After washing in TBS-T, sections were incubated for 3 minutes with 0.05% diaminobenzidine (DAB) and 0.01% H2O2 to visualise the reaction product. We skipped the primary antibodies during the staining of several of our slides to avoid non-specific binding.
Hrp conjugated goat anti rabbit secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HRP-conjugated goat anti-rabbit secondary antibody is a laboratory reagent used in immunoassays. It is a secondary antibody that binds to rabbit primary antibodies and is conjugated with horseradish peroxidase (HRP), an enzyme that can be used for signal amplification in detection systems.
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Lab products found in correlation
Goat anti mouse secondary antibody, by Thermo Fisher Scientific (3 mentions)
Whatman, by Cytiva (3 mentions)
Hrp conjugated goat anti mouse secondary antibody, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
C digit blot scanner, by LI COR (2 mentions)
Supersignal pico chemiluminescent substrate kit, by Thermo Fisher Scientific (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Phosstop phosphatase inhibitor tablet, by Roche (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Ab68672, by Abcam (1 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Bcl 2, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
39 protocols using hrp conjugated goat anti rabbit secondary antibody
1
Immunohistochemical Staining of MIF
2
Protein Extraction and Western Blot Analysis
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Frozen livers and quadriceps muscles were crushed using a mortar and pestle under liquid nitrogen and homogenized in 1 ml ice-cold 1% NP40, 1 mM EDTA in phosphate buffered saline (PBS), supplemented with complete EDTA free protease inhibitor tablets and PhosSTOP phosphatase inhibitor tablets (Roche), and centrifuged at 10,000 g for 10 min. The protein concentrations of supernatants were quantitated using the DC protein assay (Bio-Rad). Samples were resolved on 4–15% SDS-PAGE TGX Stain-Free gels (Bio-Rad) and transferred onto nitrocellulose membrane using a Trans Turbo Blot system (Bio-Rad), after imaging for total protein using the Stain-Free imaging program on the ChemiDoc MP Imaging System (Bio-Rad). Immuno-blotting was performed with rabbit polyclonal antibodies to neutrophil elastase (ab68672, Abcam), cysteine dioxygenase type 1 (ab53436, Abcam) and cysteine sulfinate decarboxylase (ab101847, Abcam) all dissolved 1:1000 in 5% bovine serum albumin (BSA). HRP-conjugated goat anti-rabbit secondary antibodies were from Thermo Fisher Scientific. Chemiluminescence signal was captured using the ChemiDoc MP Imaging System (Bio-Rad). Resultant images were quantified using ImageJ software [31 (link)]. A common sample was loaded onto each gel to normalise for detection efficiencies across membranes, and all bands were standardised to total protein for that lane [32 (link)].
3
Immunoblotting to Detect LovhK Protein
4
Western Blot Analysis of Exosome Markers and Apoptotic Proteins
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Western blotting was used to identify the U87-MG exosome markers Alix, Tsg101, CD9 (30 (link)), and Bcl-2 as well as Bax, and caspase-3 protein expression in HUVECs. Cells and exosome pellets were lysed in lysis buffer (Roche Diagnostics, Mannheim, Germany). Subsequently, 5X protein loading buffer was added directly to the protein sample, and the sample was heated at 95°C for 5 min. Next, equal amounts of protein were loaded and separated on SDS-PAGE polyacrylamide gels, and proteins were blotted onto a nitrocellulose membrane (Whatman, Maidstone, Kent, UK). Primary antibodies included Alix (mouse monoclonal anti-Alix, 1:1,000), Tsg101 (mouse monoclonal anti-Tsg101, 1:200), CD9 (rabbit monoclonal anti-CD9, 1:1,000), Bax (rabbit monoclonal anti-Bax, 1:2,000), Bcl-2 (rabbit monoclonal anti-Bcl-2, 1:1,000), and caspase-3 (mouse monoclonal anti-caspase-3, 1:500) (all from Abcam, Cambridge, UK). The primary antibodies were incubated overnight at 4°C, followed by washing and the application of HRP-conjugated goat anti-rabbit secondary antibodies (1:2,000) and goat anti-mouse secondary antibodies (1:2,000) (both from Thermo Fisher). Proteins were detected via enhanced chemiluminescence (Thermo Fisher) and imaged with a Molecular Imager VersaDoc 4000 system (Bio-Rad).
5
Immunofluorescence Imaging of ERK1/2 and p-ERK1/2
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Cells in the logarithmic growth phase were seeded on gelatin-coated coverslips. The inoculation density was 1x104 cells/ml, and the cells were allowed to adhere for 24 h and fixed with 4% PFA for 15 min at room temperature. The gelatin coating solution was purchased from Beijing Reagan Biotechnology Co., Ltd. (cat. no. IH0205). The fixed samples were washed three times with PBS for 5 min and permeabilized with 1% Triton-X-100 for 10 min. The cells were blocked with 1% bovine serum albumin (Beyotime Institute of Biotechnology; cat. no. P0007) for 1 h at room temperature and incubated with primary antibody against ERK1/2 or p-ERK1/2 (1:100 dilution) overnight at 4˚C. The samples were then washed with PBS three times and incubated with HRP conjugated goat anti-rabbit secondary antibodies (1:100 dilution; Thermo Fisher Scientific, Inc.; cat. no. 31460) for 1 h at room temperature. The nuclei were counterstained with DAPI for 15 min and the cells were washed with PBS three times for 15 min. Finally, the slides were covered with cover slips, sealed with glycerol and subjected to imaging using a fluorescence microscope (BX51; Olympus Corporation). The nuclei were visualized in blue and ERK1/2 or p-ERK1/2 were labeled with red fluorescence.
6
Histological Analysis of Femur Metaphysis
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The femur was decalcified by 10% EDTA and then embedded in paraffin. Five-micrometer sagittal sections of the metaphysis were prepared, stained with Toluidine blue, TRAP, hematoxylin-eosin (HE) and Masson, and observed by light microscopy (Zeiss, Germany) [30 (link)].
For NFATc1 and RUNX2 immunohistochemical staining, after quenching with endogenous peroxidase, achieving antigen retrieval, and blocking nonspecific binding sites, the femur section were incubated with anti-NFATc1 (1:100; Abclonal, China), anti-RUNX2 (1:100; Huabio, China) at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibodies (Invitrogen, USA) at room temperature for 30 min. Finally, the sections were exposed to diaminobenzidine peroxidase substrate for 5 min and counterstained with Mayer’s hematoxylin.
For NFATc1 and RUNX2 immunohistochemical staining, after quenching with endogenous peroxidase, achieving antigen retrieval, and blocking nonspecific binding sites, the femur section were incubated with anti-NFATc1 (1:100; Abclonal, China), anti-RUNX2 (1:100; Huabio, China) at 4 °C overnight, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibodies (Invitrogen, USA) at room temperature for 30 min. Finally, the sections were exposed to diaminobenzidine peroxidase substrate for 5 min and counterstained with Mayer’s hematoxylin.
7
ELISA for Antibody Titer Quantification
8
Western Blot Analysis of RAB27A
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One million PBMCs per donor were lysed in RIPA buffer supplemented with 1 × protease inhibitor cocktail (Santa Cruz Biotechnology) for 30 min on ice. Supernatants were mixed with 4 × NuPage loading buffer (Invitrogen) added 10 mM DTT (Invitrogen), run on a 4–12% Bis–Tris gel (Invitrogen), and transferred to a nitrocellulose membrane (iBlot, Invitrogen). Rabbit polyclonal anti-RAB27A (Proteintech Group) and HRP-conjugated goat anti-rabbit secondary antibodies (Invitrogen) were used for detection. A directly HRP-conjugated mouse anti-actin antibody (Sigma) was used as loading control. Blocking buffer and antibodies were diluted in 5% non-fat dry milk (Biorad) in TBS-Tween 0.2%.
9
Immunoblotting analysis of cell death pathways
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RIPK3 (1:2000 cat# NBP1-77299, Novus Biologicals, Centennial, CO, USA), internal control GAPDH (1:100,000, Cat# MAB374, Sigma-Aldrich, St Louis, MO, USA), β-ACTIN (1:2000, Cat# 4967, Cell Signaling Technology, Inc., Danvers, MA, USA), RIPK1(1:1000, Cat# MAB3585, R&D systems, Inc., Minneapolis, MN, USA), HIF1α (1:1000, Cat# 3716, Cell Signaling Technology, Inc., Danvers, MA, USA), cleaved CASPASE3 (1:5000, Cat# 9664, Cell Signaling Technology, Inc., Danvers, MA, USA), MLKL (1:2000, Cat# MABC604, Millipore Corp, Burlington, MA, USA), CASPASE1 (1:100, Cat# sc-56036, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and CASPASE8 (1:100, Cat# sc-81656, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) primary antibodies were used for the study. Horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (Cat# 656120, Invitrogen, Thermo Fisher Scientific, Hanover Park, IL, USA) was used for cleaved CASPASE3 (1:100,000), RIPK3, HIF1α, CASPASE8, CASPASE1 (1:20,000), goat anti-rat secondary antibody (Cat# 629320, Invitrogen, Thermo Fisher Scientific, IL, USA) was used for MLKL (1:20000) and goat anti-mouse secondary antibody (Cat# G21040, Invitrogen, Thermo Fisher Scientific, IL, USA) was used for RIPK1 (1:20,000) and GAPDH (1:100,000).
10
ZIKV Capsid Cleavage Assay
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Tartrate-purified ImmZIKV in NTE buffer was lysed in the presence of 1% DDM (Avanti) for 1 h at room temperature. To enhance viral lysis, viral samples were also sonicated for a total of 15 min. The NS2B/NS3 protease used for this assay was provided by Dr Dahai Luo (Addgene plasmid #86846; http://n2t.net/addgene:86846 ; RRID: Addgene_86846). Heat-inactivated NS2B/NS3 was prepared by pre-incubating the protease at 100 °C for 15 min and cooled down to room temperature prior use. Lysed ImmZIKV were then incubated with either NS2B/NS3 or its heat-inactivated form (0.08 mg mL−1), and RNaseA (0.1 unit μl−1) for 5 h. To look for capsid protein cleavage, the samples were then analyzed on western blotting and probed with anti-ZIKV capsid antibody (GeneTex, catalog number GTX133317) at 1:5000 dilution followed by HRP-conjugated goat anti-rabbit secondary antibody (Invitrogen, catalog number 65-6120) at 1:10,000 dilution.
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