Microtubule-associated protein 2 (MAP2) measurement was carried out using the immunocytochemistry approach to obtain the well-differentiated DRG neuron. The cell culture was fixed. The aspirated cell medium was then added 2–3 mm above the cell surface with 4% formaldehyde in 1x Phosphate buffer saline (PBS). Cells were fixed for 15 minutes at room temperature. The fixative was aspirated, then the cells were washed 3 times using 1 x PBS for 5 minutes each washing. The next stage is immunostaining. Cells were supplemented with 1x PBS blocking buffer / 5% normal serum / 0.3% Triton™ (X-100). Blocking buffer was aspirated, then Microtubule-associated protein 2 (MAP2) (GTX50810, GeneTex, USA) primary antibody was added at a 1:200 dilution in antibody dilution buffer (1x PBS / 1% BSA / 0.3% Triton) (X-100). Next, the cells were incubated overnight at 4°C. Cells were washed 3 times in 1x PBS for 5 min for each wash. Cells were incubated in goat anti-rabbit IgG secondary antibody FITC (31635, Thermo Fisher Scientific, USA) which had been diluted in antibody dilution buffer for 1–2 hours at room temperature in the dark. Cells were washed with 1x PBS 3 times for 5 minutes each wash. The cell was observed by fluorescent microscopy at the appropriate excitation wavelengths.
Goat anti rabbit igg secondary antibody fitc
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Goat anti-rabbit IgG secondary antibody FITC is a laboratory reagent used in immunoassays and other molecular biology techniques. It is a secondary antibody that binds to primary rabbit antibodies, allowing for the detection and visualization of target molecules. The antibody is conjugated with the fluorescent dye FITC (Fluorescein Isothiocyanate), enabling fluorescence-based detection methods.
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3 protocols using goat anti rabbit igg secondary antibody fitc
1
Immunocytochemistry Assay for MAP2 Quantification
2
Localization Assay for ZIKV Infection
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For localization assay, SK-N-SH and CCF-STTG1 cells were grown on glass slides with 5 × 104 cell per dish. Mock, tunicamycin-treated, and ZIKV-infected cells were designed and manipulated as described above. At 24 h post infection, the slides were washed with PBS, fixed with 4% paraformaldehyde, and then permeabilized with 0.2% Triton X-100 for 15 min. The cells were blocked by using 5% BSA and 3% normal goat serum (NGS) dissolved in PBS for 1 h at 37 °C. The cells were then washed with 3% NGS dissolved in PBS two times and then incubated in primary antibodies (1:200) diluted with 3% NGS dissolved in PBS overnight at 4 °C. After washing five times, cells were incubated with Goat anti-Rabbit IgG secondary antibody, FITC (Thermo Fisher Scientific, #F-2765), and Goat anti-Mouse IgG secondary antibody, Texas Red®-X (Thermo Fisher Scientific, #T-6390) (1:200) diluted with 3% NGS dissolved in PBS for 1 h at 37 °C. After washing two times, cell nucleus was dyed with Hoechst 33258 for 10 min at 37 °C. Microscopic analysis was performed as previously described [36 (link)]. The primary antibody used in this study was as follows: XBP1 (Abcam, ab37152), ZIKV envelope protein (BioFront Technologies, BF-1176-56).
3
Immunocytochemical Analysis of pERK
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Measurement of pERK using the immunocytochemistry approach. The differentiated DRG neuron receives treatment according to treatment groups. The treated neurons were fixed. The aspirated cell medium was then added 2–3 mm above the cell surface with 4% formaldehyde in 1x phosphate buffer saline (PBS). Cells were fixed for 15 minutes at room temperature. The fixative was aspirated, then the cells were washed 3 times using 1 x PBS for 5 minutes each washing. The next stage is immunostaining. Cells were supplemented with 1x PBS blocking buffer / 5% normal serum / 0.3% Triton™ (X-100). Blocking buffer was aspirated, then polyclonal antibody Phospho (Erk1/2) Thr202/Tyr204 (36–8800, Thermo Fisher Scientific, USA) primary antibody was added at a 1:200 dilution in antibody dilution buffer (1x PBS / 1% BSA / 0.3% Triton) (X-100). Next, the cells were incubated overnight at 4°C. Cells were washed 3 times in 1x PBS for 5 min for each wash. Cells were incubated in goat anti-rabbit IgG secondary antibody FITC (31635, Thermo Fisher Scientific, USA) which had been diluted in antibody dilution buffer for 1–2 hours at room temperature in the dark. Cells were washed with 1x PBS 3 times for 5 minutes each wash. The cell was observed by fluorescent microscopy at the appropriate excitation wavelengths.
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