The eyes were fixed in 4% PFA and transferred into 30% sucrose. Tissues were then embedded in OCT media and cut into 10-μm-thick sections using a CM1900 Cryostat (Leica, Wetzlar, Germany). Primary human astrocytes were fixed with 4% PFA for 20 mins. Then cells were permeabilized with 0.3% Triton®-X-100 PBS for 5 mins at room temperature. Sections and cells were blocked using a blocking buffer (5% donkey serum in 0.3% Triton X-100 containing PBS) for 30 mins. Primary antibodies GFAP (Abcam), RBPMS (Phosphosolutions), LCN2 (Invitrogen), 5-LOX (Abcam), Pax2 (Abcam), Vimentin (Invitrogen), and CD44 (Proteintech) were added in blocking buffer and incubated overnight at 4 °C. Sections, and cells were washed and incubated with Alexa fluor 488 and 594 conjugated secondary antibodies (Invitrogen; at 1:1000 dilution) for double labeling for 2 hrs at room temperature under dark conditions. They were then washed and mounted using ProLong™ Gold Antifade Mountant, and images were captured using a Nikon Ti Eclipse microscope.
Prolong gold antifade mountant
Manufactured by Nikon
Sourced in United States, Japan
ProLong™ Gold Antifade Mountant is a laboratory reagent used in the preparation of microscope slides. It is designed to protect fluorescent samples from fading during imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rbpms, by PhosphoSolutions (2 mentions)
5 lox, by Abcam (2 mentions)
Vimentin, by Thermo Fisher Scientific (2 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (2 mentions)
Alexa fluor 488 and 594 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Cm1900 cryostat, by Leica (2 mentions)
Sab4300061, by Novus Biologicals (2 mentions)
A1r confocal system, by Nikon (2 mentions)
Eclipse ti microscope, by Nikon (1 mentions)
A1si confocal microscope, by Nikon (1 mentions)
A1 si confocal microscopy, by Nikon (1 mentions)
7 protocols using prolong gold antifade mountant
1
Immunostaining of Human Astrocytes
2
Visualizing Osteoclast Formation and Actin Cytoskeleton
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BMMs were seeded onto coverslips of 24‐well, and RANKL was added as described above until mature osteoclasts were formed, with the presence or absence of Maackiain (40 μmol/L). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% (v/v) Triton X‐100 for 5 minutes, followed by being blocked with 3% BSA at room temperature for 10 minutes. The cells were incubated with anti‐vinculin or anti‐NFATc1 antibody at 4°C, followed by the incubation with a secondary anti‐mouse IgG conjugated with FITC (Thermo Fisher). Then, the cells were co‐stained with rhodamine phalloidin and Hoechst 33258 for 20 minutes. After the staining, the coverslips were mounted with the ProLong Gold Antifade Mountant and visualized using a confocal microscope (Nikon Corporation) as previously described.27
3
Cryosections Immunostaining of ErbB2
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Cryosections were fixed in 4% PFA, permeabilized with 0.2% Triton X‐100, subjected to antigen retrieval (2N HCL), and blocked in 1% BSA/10% donkey serum. Samples were then incubated overnight at 4°C with primary antibodies against ErbB2 (1 : 100; R&D Systems, #AF5176, Minneapolis, MN, USA), or phosphorylated ErbB2 (Tyr1248) (1 : 50; Sigma‐Aldrich, #SAB4300061, St. Louis, MO, USA), and co‐stained with anti‐neurofilament (1 : 5000; Novus, #NB300‐217, Littleton, CO, USA) and anti‐myosin heavy chain (1 : 100; DSHB, clone S46). [11] Following 1 h of incubation with a secondary Alexa Fluor antibody (1 : 200–800; Invitrogen, Waltham, MA, USA), slides were mounted with ProLong™ Gold Antifade Mountant and visualized on a Nikon A1R Confocal System.
4
Osteoclast Formation and Cytoskeletal Analysis
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5 × 104 BMMs were seeded onto glass coverslips (14 mm in diameter) in 24‐well plates per well. The BMMs were then induced to form mature osteoclasts, as described previously, in the presence or absence of D.P (30 μmol/L). The cells were then fixed and permeabilized with 0.1% (v/v) Triton X‐100. After blocking unspecific binding using 3% BSA in PBS, the cells were incubated with anti‐vinculin antibody (1:500) or anti‐NFATc1 antibody (1:500) at 4°C overnight. A secondary antimouse IgG conjugated with FITC was used to produce a fluorescent signal. After the incubation of secondary antibody for 1 hour, the cells were stained with rhodamine phalloidin for 20 minutes. Last, the cells were incubated with DAPI or Hoechst 33258 dye for 10 minutes and mounted to glass slides using the ProLong Gold Antifade Mountant and visualized using NIKON A1Si confocal microscope (Nikon Corporation).
5
Immunostaining of Human Astrocytes
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The eyes were fixed in 4% PFA and transferred into 30% sucrose. Tissues were then embedded in OCT media and cut into 10-µm-thick sections using a CM1900 Cryostat (Leica, Wetzlar, Germany). Primary human astrocytes were fixed with 4% PFA for 20 min. The cells were permeabilized with 0.3% Triton®-X-100 PBS for 5 min at room temperature. Sections and cells were blocked using a blocking buffer (5% donkey serum in 0.3% Triton X-100 containing PBS) for 30 min. Primary antibodies against GFAP (Abcam), RBPMS (Phosphosolutions), LCN2 (Invitrogen), 5-LOX (Abcam), Pax2 (Abcam), Vimentin (Invitrogen), and CD44 (Proteintech) were added to blocking buffer and incubated overnight at 4 °C. Sections and cells were washed and incubated with Alexa Fluor 488- and 594- conjugated secondary antibodies (Invitrogen; at 1:1000 dilution) for double labeling for 2 h at room temperature under dark conditions. They were then washed and mounted using ProLong™ Gold Antifade Mountant, and images were captured using a Nikon Ti Eclipse microscope.
6
Immunolabeling of Phosphorylated ErbB2 in Tissue Sections
7
Visualization of Osteoclast Actin Rings
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To observe actin rings, osteoclasts were induced as above and were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% (v/v) Triton X-100 for 10 minutes, blocked with 3% bovine serum albumin (BSA), and subsequently stained with Rhodamine Phalloidin (1:300 dilution) in the dark for 2 h. Cells were then washed with PBS twice, followed by incubation with DAPI for 10 minutes to visualize nuclei. After staining, cells were mounted in ProLong Gold Antifade Mountant and observed under NIKON A1Si confocal microscopy (Nikon Corporation, Tokyo, Japan). Three images were randomly captured for each group and the number of nuclei and the area of the actin rings were analysed using Image J software.
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