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7 protocols using bs 0296g fitc

1

Immunofluorescence Staining Protocol for KDM2A, FABP4, Ki67, and PPARγ

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For immunofluorescence staining, the cells were fixed with 4% PFA (Biosharp, BL539A, China) for 10–15 min, washed three times with PBS and then incubated with 100 mM glycine for 5–10 min to terminate the PFA. A block solution containing 5% goat serum, 2% BSA, 0.2% Triton X-100 and 0.1% sodium azide PBS solution was closed at room temperature for 30 min. Cells were incubated with a primary anti-KDM2A antibody (Abcam, EPR18602, UK), mouse anti-FABP4 antibody (Bioss, bsm-51247M, Shanghai, China), anti-Ki67 rabbit pAb (Wanlei, WL01384a, Shenyang, China) and anti-PPARγ rabbit pAb (Wanlei, WL01800, China) at 1:500 dilution in a blocked solution at 4 °C overnight. Alexa FluorTM 568 goat anti-rabbit igG (Invitrogen, A11011, Carlsbad, CA, USA) and goat anti-mouse IgG/FITC (Bioss, bs-0296G-FITC, Shanghai, China) conjugated secondary antibodies were used at a dilution of 1:500 and 1:1000 at room temperature for 1 h, respectively. All the pictures were taken under a Zeiss confocal microscope.
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2

Immunofluorescent Analysis of NSC Differentiation

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Cells were fixed with 4% paraformaldehyde for 15-30 min at room temperature. After washing with PBS for three times, the cells were covered with 4% BSA solution containing 0.1% Triton X-100 at room temperature for 1 h and then incubated overnight with 4% BSA-diluted primary antibodies β-tubulin III (1:500, ab215037; Abcam) and GFAP (ab279290, 1:50; Abcam). The samples were removed from the 4˚C condition on the second day, reheated at room temperature for 30 min and then incubated with goat anti-rabbit IgG Cy5 (1:500, bs-0295G-Cy5; Bioss) or goat anti-mouse IgG FITC (1:500, bs-0296G-FITC; Bioss) at room temperature for 1 h. The solution was then discarded, and the samples were washed with PBS for three times. Nuclei were stained at room temperature with Hoechst 33342 (C0030; Beijing Solarbio Science & Technology Co., Ltd.). The NSCs differentiation were identified by detecting the expression of β-tubulin III and GFAP under a fluorescence microscope (scale bar, 20 µm; Zeiss AG).
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3

Porcine PMEC PRRSV Infection and Receptor Detection

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Porcine PMECs (4th–5th passage) were seeded into 24-well plates for immunofluorescence. To detect viral antigens, cells were incubated for 12 hr and then
infected with the PRRSV strain HN. Immunofluorescence staining for PRRSV GP5 and N proteins was carried out at 48 hpi using polyclonal antibodies (Cat #bs-4504R
and Cat #23941R, Bioss, Beijing, China). To detect the receptors of PRRSV, the PMECs were fixed with 4% paraformaldehyde after a 24-hr incubation, and a routine
indirect immunofluorescence method was performed. Anti-CD151 (Cat #sc-18753-R, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD163 (Cat #MCA2311GA, AbD
Serotec, Kidlington, Oxford, UK), and anti-CD169 (Cat #MCA2316GA, AbD Serotec) antibodies and goat anti-mouse (Cat #bs-0296G-FITC, Bioss) and goat anti-rabbit
(Cat #bs-0296G-FITC, Bioss) FITC-conjugated secondary antibodies were used.
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4

Immunocytochemical Characterization of Human Tenocytes

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Immunocytochemical staining was performed in vitro to characterize human primary tenocytes following a standard protocol [11 ], using antibodies against COL1 (1:200), COL3 (1:200), vimentin (1:200), SCX (1:400), CD34 (1:200), and OCT4 (1:200). When using mouse primary antibodies, normal goat serum (Solarbio; A8020) was used for blocking and a goat anti-mouse secondary antibody conjugated with fluorescein isothiocyanate (FITC) (Bioss; bs-0296G-FITC) was applied. For rabbit primary antibodies, normal goat serum (Solarbio) and a goat anti-rabbit secondary antibody (Bioss; bs-0295G-AF594) were used. Cell nuclei were counterstained with DAPI-containing mounting media (Solarbio; S2110) and analyzed under a fluorescence microscope (Olympus BX53; Olympus Corporation, Tokyo, Japan). The montages were created using Adobe Photoshop (version CC2017; Adobe, San Jose, CA, USA).
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5

Immunofluorescence Analysis of Isolated CAFs and NFs

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The isolated CAFs or NFs were fixed in 4% paraformaldehyde. After permeabilization in 0.2% Triton X-100 and blocking in goat serum, the cells were probed with primary antibodies α-SMA (1:500, bsm-33187M, Bioss, Beijing, China) and vimentin (1:100, bsm-0756R, Bioss) overnight at 4°C. Subsequently, Goat Anti-Mouse IgG/FITC (1:100, bs-0296G-FITC, Bioss) and Goat Anti-rabbit IgG/Cy3 (1:100, bs-0295G-Cy3, Bioss) were applied. After nuclear counterstaining with DAPI, the cells were observed using a fluorescent microscope (Olympus, Tokyo, Japan).
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Immunofluorescence Staining of Brain Sections

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Para n-embedded brain sections were rinsed in 0.01 M PBS (pH 7.3) 3 times (10 min for each) and blocked in 0.01 M PBS containing 10% normal donkey serum and 0.3% Triton X-100 for 1 h at RT. And then, the sections were incubated for 1 h at RT and for 48 h at 4°C with primary antibodies: goat anti-Iba1 antibody (1:500; ab5076, abcam, USA), mouse anti-GFAP (1:1000; MAB3402, Merck, Germany), mouse anti-NeuN (1:1000; MAB377, Merck, Germany) and rabbit anti-NEK7 antibody (1:800; NBP1-31110, NOVUS, USA) in PBS containing 0.3% (v/v) Triton X-100, 0.25% (w/v) λ-carrageenan, and 5% (v/v) donkey serum (PBS-XCD). All sections were washed three times in 0.01 M PBS (10 min each), and were then incubated for 1.5 h at RT with Donkey Anti-Goat IgG/FITC antibody (1:1000; bs-0294D-FITC, BIOSS, China), Donkey Anti-rabbit IgG/Cy3 antibody (1:1000; bs-0295D-Cy3, BIOSS, China) and Goat Anti-Mouse IgG/FITC antibody (1:1000; bs-0296G-FITC, BIOSS, China) respectively. Finally, all sections were air-dried and coverslipped with a mixture of 0.05 M PBS containing 50% (v/v) glycerin and 2.5% (w/v) triethylenediamine. The confocal images were obtained, and digital images were captured using a Fluoview laser scanning confocal microscopes (Olympus) equipped with the FV1000 (Ver.1.7a) software.
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7

Immunofluorescence Staining of Brain Sections

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Para n-embedded brain sections were rinsed in 0.01 M PBS (pH 7.3) 3 times (10 min for each) and blocked in 0.01 M PBS containing 10% normal donkey serum and 0.3% Triton X-100 for 1 h at RT. And then, the sections were incubated for 1 h at RT and for 48 h at 4°C with primary antibodies: goat anti-Iba1 antibody (1:500; ab5076, abcam, USA), mouse anti-GFAP (1:1000; MAB3402, Merck, Germany), mouse anti-NeuN (1:1000; MAB377, Merck, Germany) and rabbit anti-NEK7 antibody (1:800; NBP1-31110, NOVUS, USA) in PBS containing 0.3% (v/v) Triton X-100, 0.25% (w/v) λ-carrageenan, and 5% (v/v) donkey serum (PBS-XCD). All sections were washed three times in 0.01 M PBS (10 min each), and were then incubated for 1.5 h at RT with Donkey Anti-Goat IgG/FITC antibody (1:1000; bs-0294D-FITC, BIOSS, China), Donkey Anti-rabbit IgG/Cy3 antibody (1:1000; bs-0295D-Cy3, BIOSS, China) and Goat Anti-Mouse IgG/FITC antibody (1:1000; bs-0296G-FITC, BIOSS, China) respectively. Finally, all sections were air-dried and coverslipped with a mixture of 0.05 M PBS containing 50% (v/v) glycerin and 2.5% (w/v) triethylenediamine. The confocal images were obtained, and digital images were captured using a Fluoview laser scanning confocal microscopes (Olympus) equipped with the FV1000 (Ver.1.7a) software.
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