Embryonic brains were dissected from E14.5 litters obtained from pregnant mice. Coronal brain sections, 10–20 μm thick, were cut on a cryostat and mounted directly onto poly-l -lysine-coated slides. The sections were air-dried and subsequently fixed with 50% ice-cold methanol–acetone mixture for 10 min at −20°C. After being blocked with 10% goat serum, sections were incubated with anti-Nestin (1:500; Novus Biologicals) and anti-C1qR1 (1:200; Millipore) overnight at 4°C. Preincubation with a 10-fold excess of specific blocking peptide (1st BASE) against C1qR1 antibody was used as a negative control for this reaction. The following day, the sections were washed several times with PBS and incubated with anti-chicken Alexa Fluor 488 IgG and anti-mouse Alexa Fluor 568 IgG (1:1,000; Molecular Probes) secondary antibodies for 1 h at room temperature. The sections were counterstained with DAPI (Invitrogen). Fluorescent mounting media were applied before placing coverslips onto the slides. The cells immunoreactive for C1qR1 and Nestin were examined under an Olympus FV1000 confocal microscope.
Anti nestin
Manufactured by Novus Biologicals
Sourced in China
Anti-Nestin is a lab equipment product that can be used to detect the expression of the Nestin protein, which is commonly used as a marker for neural stem cells and progenitor cells. It is a specific antibody that binds to the Nestin protein, allowing for its identification and quantification in various experimental systems.
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Lab products found in correlation
Evos fl auto imaging system, by Thermo Fisher Scientific (2 mentions)
Anti βiii tubulin, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Anti sox2, by R&D Systems (1 mentions)
Ba 9500, by Vector Laboratories (1 mentions)
Ba 9010, by Vector Laboratories (1 mentions)
Anti gfap, by Abcam (1 mentions)
Anti ghrh, by Cloud-Clone (1 mentions)
Anti trh, by Cloud-Clone (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor r 594, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti nestin
1
Immunofluorescence analysis of Nestin and C1qR1 in embryonic mouse brains
2
Immunohistochemical Analysis of Neural Markers
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All samples were wax embedded and processed as previously reported69 (link). The following antibodies and dilutions were used: Anti-Sox2 (R&D cat# af2018) at 1:150, Anti-Nestin (Novus cat# NB100-1604) at 1:500 and Anti-pdgfra (R&D cat# af1062) at 1:20, Anti-Goat IgG (H + L), made in horse, biotinylated (Vector labs cat# BA-9500) at 1:200 and Anti-chicken IgG (H + L), made in goat, biotinylated (Vector labs cat#BA-9010) at 1:200 dilution.
3
Immunofluorescence Staining of Cell Markers
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For immunofluorescence, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100/PBS for 15 min each at room temperature. After blocking samples with 2% BSA/PBS for 30 min, cells were subjected to immunofluorescence staining with anti-Gfap (1:1000, abcam), anti-Nestin (1:100, Novus Biologicals), anti-phosphor-Histone3 at Ser 10 (1:200, Cell Signaling), and anti-βIII tubulin (1:200, abcam) primary antibodies overnight at 4 °C. Next day, cells were washed with cold PBS and incubated with Flamma®552- or Flamma®488- conjugated goat anti-rabbit IgG (bioacts) or goat anti-mouse IgG (bioacts, Incheon, Korea) for 30 min at room temperature. Fluorescence signals were visualized with EVOS FL Auto Imaging System (thermo fisher scientific).
4
Immunofluorescence Staining of Neural Markers
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Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100/PBS for 15 min each at room temperature. After blocking samples with 2% BSA/PBS for 30 min, cells were subjected to immunofluorescence staining with anti-Nestin (1 : 100, Novus Biologicals), anti-FoxO1 (1 : 200, Thermo Fisher Scientific), and anti-βIII tubulin (1 : 200, Abcam) primary antibodies overnight at 4°C. The next day, cells were washed with PBS and incubated with Flamma®552- or Flamma®488-conjugated goat antirabbit IgG (Bioacts) or goat antimouse IgG (Bioacts) for 30 min at room temperature. Fluorescence signals were visualized with the EVOS FL Auto Imaging System (Thermo Fisher Scientific).
5
Immunohistochemical Analysis of ZIKV Infection
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The hypothalamus collected from ZIKV-infected or control mice was embedded immediately in optimal cutting temperature compound (OCT, SAKURA, USA) to prepare frozen sections (6 mm in thickness). The sections were incubated with mouse anti-ZIKV polyclonal antibody (PAb) (1:200), anti-GHRH (1:100, Cloud-Clone,China), anti-TRH (1:100, Cloud-Clone, China), or anti-Nestin (1:100, novus biological, USA) as primary antibody at 4 C overnight, and then incubated with goat anti-mouse IgG (1:400, Alexa Fluor 488, Life technologies, USA) or donkey anti-rabbit IgG (Alexa Fluor R 594, Life technologies, USA) at 37 C for 1 h. All images were captured with an Olympus microscope (IX71, Olympus, Japan).
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