Cells were fixed with 4% w/v formaldehyde in 1x PBS for 20 min and permeabilised with 0.25% v/v Triton-X in 1x PBS for 20 min. HBV core and surface antigens were detected using a 1:3,000 dilution of polyclonal rabbit anti-HBcAg antibody (B0586, Dako)29 (link) and a 1:3,000 dilution of HBsAb (Humabs Biomed, kind gift from Davide Corti) in 1x PBS and 2% w/v IgG-free bovine serum albumin (Sigma Aldrich) overnight at 4°C. After washing with PBS, cells were overlaid with 1:500 AF488-conjugated goat anti-rabbit secondary antibody (A-11008, Invitrogen, Carlsbad, CA, USA), 1:1,000 AF555-conjugated goat anti-human secondary antibody (A-21433, Invitrogen), and 2 μg/ml Hoechst 33342 (H1399, Invitrogen) in 1x PBS and 2% w/v IgG-free bovine serum albumin (Sigma Aldrich) and then incubated in the dark for 1 h. Fluorescence microscopy images were acquired at 20x magnification. For each sample, an area the size of 5 × 5 fields of view were acquired and merged by NIS Elements Advanced software (Nikon). Images were edited and analysed with Fiji ImageJ imaging software.34 (link)
Igg free bovine serum albumin
Manufactured by Merck Group
Sourced in Germany, United States
IgG-free bovine serum albumin is a laboratory reagent used as a protein stabilizer and blocking agent in various immunoassay and cell culture applications. It is purified from bovine serum to remove immunoglobulin G (IgG) content, making it suitable for use in sensitive immunological techniques where IgG interference must be avoided.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescence mounting medium, by Agilent Technologies (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Goat serum, by Merck Group (2 mentions)
Daf fm diacetate, by Thermo Fisher Scientific (2 mentions)
Anti c4d monoclonal antibody, by Quidel (2 mentions)
Igg1k isotype control, by BioLegend (2 mentions)
Hanks balanced salt solution with ca2 and mg2 hbss, by Thermo Fisher Scientific (2 mentions)
Chemiluminescence method, by GE Healthcare (1 mentions)
Lamin a c, by Cell Signaling Technology (1 mentions)
Propidium iodide, by Vector Laboratories (1 mentions)
Sc 47089, by Santa Cruz Biotechnology (1 mentions)
Tcp sp2, by Leica (1 mentions)
Cm1100, by Leica (1 mentions)
B0586, by Agilent Technologies (1 mentions)
Nis elements advanced software, by Nikon (1 mentions)
Af488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Dylight 488, by Merck Group (1 mentions)
Eosin 5 maleimide, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated rabbit anti mouse igg, by Thermo Fisher Scientific (1 mentions)
7 protocols using igg free bovine serum albumin
1
Immunofluorescence Staining of HBV Antigens
2
Isolation and Fractionation of Cellular Compartments
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Cytoplasmic and nuclear fractions were prepared using our established method [28 (link)]. TS (Triton-X-100-soluble) fraction and TI (Triton-X-100 insoluble, but n-octyl-β-D-glucoside soluble) fraction—TI fraction by definition, represents lipid raft—were isolated according to the established protocol [7 (link), 29 (link)]. Presence of ectopic HA-tagged STK4 protein in cytoplasmic, lipid raft and nuclear fractions was determined by Western blotting using the HA antibody (Covance). Briefly, proteins were resolved by SDS-PAGE. PBST (0.1% Tween-20) containing 5% (w/v) skim milk powder or PBST containing 5% immunoglobulin G (IgG)-free bovine serum albumin (Sigma-Aldrich) was used in membrane blocking and antibody dilutions. Giα2 protein was included as a negative and positive control for TS and TI fractionations, respectively. Lamin A/C (Cell Signaling Technology) was used as a negative and positive control for cytoplasmic and nuclear fractionations, respectively [27 (link)]. Signals were visualized by chemiluminescence method (GE HealthCare).
3
Macroscopic Analysis of OA-Induced Articular Cartilage
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To analyse the macroscopic characteristics of articular cartilage, femoral condyles were obtained from normal and OA-induced rats (3, 5, and 10 days) and observed under a stereomicroscope (E2 4D Leica, Heidelberg, Germany). The cartilage samples were then cryopreserved with 10% PBS-sucrose for 24 h, cryosectioned (Leica CM 1100; Heerbrugg, Switzerland), mounted on gelatine-coated slides, and stored at −20°C for 2 days. The samples were hydrated in PBS, treated with 0.2% Tween 20 in PBS for 10 min, and preincubated with 0.2% IgG-free bovine serum albumin (Sigma Chemical, Germany) for 20 min at room temperature. The sections were incubated overnight at 4°C with anti-Lxn goat polyclonal antibody (1:70, sc-47089, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by incubation with fluorescein isothiocyanate (FITC)-tagged anti-goat IgG (1:50 Zymed Laboratories, South San Francisco, CA) for 1 h at room temperature. The nuclei were counterstained with propidium iodide for 1 min (1:3000; Vector Laboratories, Burlingame, CA), and the samples were mounted with Vectashield. As a negative control, the primary antibody was omitted. Rat heart tissue was used as a positive control. The sections were analysed with confocal microscopy (TCP-SP2, Leica, Heidelberg, Germany). These experiments were performed in triplicate.
4
Immunofluorescence Staining Protocol
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Sections were washed in phosphatebuffered saline (PBS) for 5 min and then fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA, USA) for 10 min at room temperature. All washes (3x10 min) between stages were performed in PBS. After the sections had been permeabilized with 0.2% Triton X-100 (Sigma) in PBS for 5 min, potential nonspecific binding sites were blocked with antibody dilution buffer (2% goat serum (Sigma) and 1% IgG-free bovine serum albumin (Sigma) in PBS) for 20 min at room temperature. Sections were then incubated with primary antibodies overnight at 4°C. In negative control experiments, primary antibodies were omitted. After being washed, the sections were then incubated with secondary antibodies for 1 h at room temperature. Following the final washing step, the glass coverslips were mounted upside down on clean microscope slides with Fluorescence Mounting Medium (DAKO).
5
Sequential Immunostaining of GFAP and p75NTR
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Sections were fixed in methanol at 4°C for 3 min, and then in acetone at 4°C for 3 min. All the following steps were performed at room temperature. The sections were airdried for 1 h and then rehydrated for 10 min in phosphate-buffered saline (PBS). All washes (3x10 min) between stages were performed in PBS. After the sections had been permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 5 min, potential non-specific binding sites were blocked with antibody dilution buffer [2% goat serum (Sigma-Aldrich) and 1% IgG-free bovine serum albumin (Sigma-Aldrich) in PBS] for 20 min at room temperature. Sections were then incubated with primary antibodies overnight at 4°C. After being washed, the sections were then incubated with secondary antibodies for 1 hour at room temperature. Following the final washing step, sections were mounted with Fluorescence Mounting Medium (Dako, North Sydney, Australia).
For immunostaining with two rabbit antibodies [anti-glial fibrillary acidic protein (GFAP) and anti-p75 neurotrophin receptor (p75NTR)] on the same section, a sequential immunostaining approach30 (link) was used [anti-GFAP+goat anti-rabbit IgG DyLight 488+ rabbit IgG (Sigma) + (mixture of anti-p75NTR +goat anti-rabbit IgG Alexa 555; incubated half hour before application)].
For immunostaining with two rabbit antibodies [anti-glial fibrillary acidic protein (GFAP) and anti-p75 neurotrophin receptor (p75NTR)] on the same section, a sequential immunostaining approach30 (link) was used [anti-GFAP+goat anti-rabbit IgG DyLight 488+ rabbit IgG (Sigma) + (mixture of anti-p75NTR +goat anti-rabbit IgG Alexa 555; incubated half hour before application)].
6
Immunofluorescence Staining of Complement Proteins
7
Immunofluorescence Staining for C4d
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