Cells were grown on coverslips overnight. Then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in warm PBS for 15 min in room temperature. Then aspirate fixative, rinse three times in 1 × PBS for 5 min each. After treating with 0.5% Triton-X100 (ThermoFisher, USA) diluted in warm PBS and blocking specimen in blocking buffer (5% Bovine Serum Albumin diluted in warm PBS, BioFroxx, Guangzhou, China) for 60 min in room temperature, primary antibodies were applied for 4 C overnight. AlexaFluor488- conjugated anti-mouse IgG (1:1000, Molecular Probes, A11029), AlexaFluor488-conjugated anti-rabbit IgG (1:1000, Molecular Probes, A11034) or AlexaFluor594-conjugated anti-rabbit IgG (1:1000, Molecular Probes, A11037) secondary antibodies were used in room-temperature for 1 h. Nuclei were counterstained with DAPI (1 μg/ml, Molecular Probes, D1306). Protein subcellular localization was analyzed under a Zeiss 510 META or Leica TCS-SP2 confocal laser scanning microscope. The concentration of primary antibody was rabbit anti-CD68 (1:200, Abcam, ab125212), rabbit anti-CD163 (1:200, Proteintech, 16,646–1-AP), rabbit anti-CD204 (1:200, Abcam, ab123946), rabbit anti-CD206 (1:200, Abcam, ab64693), mouse anti-Arg1 (1:200, Abcam, ab239731), rabbit anti-Mrc1 (1:200, Abcam, ab64693), rabbit anti-Fizz1 (1:200, Abcam, ab39626).
510 meta
Manufactured by Leica
The 510 META is a versatile laser scanning confocal microscope designed for high-resolution imaging. It features a multi-wavelength laser system and advanced optics to provide detailed analysis of samples.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by neoFroxx (2 mentions)
Triton x 100, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab123946, by Abcam (1 mentions)
Ab239731, by Abcam (1 mentions)
Ab39626, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Alexa fluor 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Tcs sp2, by Leica (1 mentions)
Eclipse te200, by Zeiss (1 mentions)
Prolong gold containing 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
3 protocols using 510 meta
1
Immunofluorescence Analysis of Macrophage Markers
2
Immunocytochemistry of BDNF-treated Cells
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Cultured cells were treated with BDNF for the indicated times, washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were permeabilized with PBS/0.5%Triton X-100 and then blocked for 1 hr with PBS/1% BSA/5% normal goat serum and exposed to primary antibodies overnight at 4°C. Cells were washed 3X with PBS, and exposed to secondary antibodies coupled to different fluorophores for 1 hr at room temp. Cells were washed three times in PBS and then mounted using Prolong Gold containing 4′,6′-diamidino-2-phenylindole (DAPI) (Life Technologies P36934). Cells were analyzed by epifluorescence (Nikon Eclipse TE200), confocal (Zeiss 510 Meta), or enhanced resolution (Leica SP8, 63X, 1.4 NA, Lightning Mode) microscopy. No immunostaining was seen in controls with omission of the primary antibodies.
3
Immunofluorescence Staining and Confocal Microscopy
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Cells were grown overnight on coverslips and then covered to a depth of 2-3 mm with 4% formaldehyde diluted in 1 × PBS for 15 min in room temperature. Then the fixative was aspirated, followed by rinsing thrice, each for 5 min, in 1 × PBS. After treating with 0.5% Triton-X100 (ThermoFisher, USA) diluted in warm PBS and blocking agent in blocking buffer (5% bovine serum albumin diluted in warm PBS, BioFroxx, Guangzhou, China) for 1 h at room temperature, the cells were incubated with primary antibodies overnight at 4 °C followed by secondary antibodies for 1 h at room temperature. The primary and secondary antibodies used are shown in Table S1 . Nuclear staining was performed using 1 μg/mL of 4'-6-diamidino-2-phenylindole (DAPI, Molecular Probes, D1306). Protein subcellular localization was observed under a Zeiss 510 META or Leica TCS-SP2 confocal laser scanning microscope.
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