Cells were fixed first with 2% paraformaldehyde for 5 min and then with cold methanol (−20°C) for 20 min. Cells were then washed with PBS, blocked with 5% goat serum, and incubated overnight at 4°C with primary antibodies (1:100) against HIV-1 Tat (Dr. Tory Johnson), HA (Abcam), GM130 (Abcam), and LAMP1 (Cell technology). After washing with PBS, cells were incubated with corresponding Alexa 488- or Alexa 647-conjugated secondary antibodies (Invitrogen). Cells were examined by confocal microscopy (Zeiss LSM800). Controls for immunostaining specificity included staining cells with primary antibodies without fluorescence-conjugated secondary antibodies (background controls), and staining cells or rat neurons with only secondary antibodies; these controls helped eliminate auto-fluorescence in each channel and bleed-through (crossover) between channels.
Alexa 488 or alexa 647 conjugated secondary antibodies
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488- or Alexa Fluor 647-conjugated secondary antibodies are fluorescent-labeled antibodies that can be used to detect and visualize target proteins in various applications, such as Western blotting, immunohistochemistry, and flow cytometry. The Alexa Fluor dyes provide bright, photostable fluorescence to enable sensitive detection of the target proteins.
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Lab products found in correlation
4 protocols using alexa 488 or alexa 647 conjugated secondary antibodies
1
Immunostaining of HIV-1 Tat Protein
2
Immunofluorescent Labelling of DARPP32 and NRG4
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Brains were fixed overnight using 4% paraformaldehyde in 0.12 M PBS at 4°C, washed in PBS and cryoprotected in 30% sucrose before being frozen in dry ice-cooled isopentane. Serial 30 μm sections were blocked in 1% bovine serum albumin (Sigma), 0.1% Triton (Sigma) in PBS and then incubated with 1:400 rabbit-monoclonal anti-DARPP32 (Cell Signaling) and 1:200 goat-polyclonal anti-NRG4 (Santa Cruz Biotechnologies, Santa Cruz, CA) antibodies at 4°C overnight. After washing, the sections were incubated with 1:500 of donkey anti-rabbit or goat-polyclonal Alexa-488 or Alexa-647 conjugated secondary antibodies (Invitrogen) for 1 hour at room temperature. Sections were washed, incubated with DAPI, and visualized using a Zeiss LSM710 confocal microscope.
3
Immunofluorescence Staining Protocol
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Cells were fixed in 4% paraformaldehyde in PBS for 10 min, permeabilized for 10 min in 0.1% Triton X-100, and blocked for 1 h in 2% BSA. The cells were then incubated in primary antibody overnight, washed with PBS (3 × 5 min), incubated with the secondary antibody in 2% BSA for 1 h, washed (3 × 10 min) and stained with 1 μg ml−1 DAPI for 10 min. Mounting media was composed of 2% of n-propyl gallate in 90% Glycerol and 10% PBS. Analysis was done on a Leica TCS-SPE Confocal microscope using a ×40 objective and Leica Software. The antibodies used for immunostaining were anti-TFE3 (Sigma Prestige HPA023881, 1:400), anti-Oct4 (Santa Cruz sc-5279, 1:100), and Alexa 488- or Alexa 647-conjugated secondary antibodies (Molecular Probes).
4
Immunohistochemistry of Mouse Brain Sections
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Mice were anaesthetized and perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). The brains were removed, postfixed overnight at 4 °C and sectioned at a thickness of 50 μm. Free-floating sections were incubated in blocking solution (PBS containing 10% normal sheep serum and 0.1% Triton X-100) overnight, followed by incubation with primary antibodies(diuluted 1 to 500) for 3 days and, after extensive washing, with Alexa 488- or Alexa 647-conjugated secondary antibodies (Molecular Probes) overnight at 4 °C.
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