Fluorescein isothiocyanate (fitc)

Cells were seeded onto coverslips in 24-well plates and incubated overnight to achieve 75–80% confluency. For IFA, cells were seeded at the following densities: 4×10⁴ cells/well for SJCRH30, 8×10⁴ cells/well for SK-N-SH, 7×10⁴ cells/well for HeLa CCL2, 4×10⁴ cells/ml, 7×10⁴ cells/ml of C2C12 and 2x10⁵ cells/well for C6/36. Cell monolayers were infected with CHIKV-0708 at a multiplicity of Infection (MOI) of 10 for 1 hour at 37°C to allow viral attachment and entry. Infected cells were washed twice with PBS and topped up with maintenance media to allow virus growth. Cells were then incubated for 24 h.p.i before fixation. Supernatants were removed and cells were washed once with PBS prior to fixation in ice-cold methanol for 15 min. This was followed by three washes of cold PBS prior to incubation with primary antibodies. The cells were then subjected to immunofluorescence staining using appropriate antibodies and/or fluorescent dyes, such as primary anti-alphavirus mouse monoclonal antibodies (Santa Cruz Biotechnology, 1:100 dilution), followed by secondary goat anti-mouse IgG conjugated to fluorescein isothiocyanate (FITC; Invitrogen, 1:500 dilution) antibody. Cell nuclei were counter-stained with DAPI (Sigma Aldrich, Duolink in situ mounting media). Coverslips were then viewed at DAPI and FITC channels at 100× magnification (Olympus IX81, inverted microscope) [24 (link)].

HSMM cell monolayers grown overnight on coverslips till 75% confluency were infected with CHIKV strain Singapore 072008 at an MOI of 10 for 2 hrs at 37 °C. Infected cells were fixed in ice-cold absolute methanol and washed three times with cold PBS. The cells were then subjected to immunofluorescence staining using primary customised rabbit polyclonal antibodies targeted against CHIKV E2 protein (ProSci Inc., USA) at a 1:100 dilution, followed by secondary antibodies conjugated to fluorescein isothiocyanate (FITC; Invitrogen) at a 1:1000 dilution. Cell nuclei were counter-stained with 4′, 6′-diamidino-2-phenylindole (DAPI; 100 nM) fluorescent dye (Invitrogen) at a 1:250,000 dilution. The specimens were then viewed with Olympus IX81 motorized inverted epifluorescence microscope (Olympus, Japan) with appropriate excitation and emission wavelengths for FITC (490 nm and 525 nm respectively), and DAPI (350 nm and 470 nm respectively) at 100x and 1000x magnification.

Neurobiotin (2.5%) was loaded into the BC soma of Tg(Gal4-VP16^xfz43,UAS:nfsB-mCherry) larvae via whole-cell patch-clamp recording micropipettes for >30 min. Due to the small size of zebrafish BCs, the soma of recorded cells was often pulled away by the removal of micropipettes. After the recording, the larvae were incubated in 4% paraformaldehyde (PFA) overnight at 4 ^oC. At the next day, the larvae were first rinsed with 0.1 M PBST (PBS with 0.5% Tween) three times at the interval of 30 min, and then incubated in 0.1 M PBS containing 1:200 strepetavidin-conjugated FITC (Invitrogen) for 5 h at room temperature. After rinsed with 0.1 M PBST three times at the interval of 30 min, the tissue was embedded in 1.2% low melting-point agarose for the following imaging with Olympus FV1000 confocal microscope (excitation wavelength: 488 nm for FITC, 559 nm for mCherry).

Immunofluorescence was performed as described by Dong et al. (2016 (link)). After deparaffinization and washing, the preincubated sections were incubated with the mouse monoclonal antibody anti-Calbindin-D-28 K (Sigma-Aldrich, St. Louis, MO, USA; ratio of 1:3000) and the rabbit polyclonal antibody anti-GAD65 (Santa Cruz Biotechnology, Inc., USA; ratio of 1:50), or the mouse monoclonal antibody anti-Calbindin-D-28 K and the rabbit polyclonal antibody anti-Neurofascin (Santa Cruz Biotechnology, Inc., USA; ratio of 1:25) for double immunofluorescence labeling. Then, tissue sections were washed in PBS after overnight at 4°C, and incubated for 2 h using secondary antibody conjugated to the fluorescent markers FITC and Rhodamine (Zhongshan Biotechnology, Beijing, China; ratio of 1:100). Finally, tissue sections were mounted with glycerin gelatin for histological examination. The slices labeled by FITC and Rhodamine were observed by a fluorescence microscope (BX61+DP-71; Olympus/IPP, Japan/USA). And these images were obtained from cerebellar lobule 4–5 and then merged at a magnification of × 400 (ocular × 10 and objective × 40), respectively. The mean intensities of GAD65 and NF186 in the cerebellum were obtained by using image analysis program (MetaMorph, UIC, US). Three different fields per section were selected, and three sections per animal were measured to get a mean value.

The calcium imaging was performed as previously described [27 (link)]. The cells were seeded in a 3.5 cm dish containing glass coverslips for 24 h and loaded with 10 μM Fluo-4-AM (Dojindo, Kumamoto, Japan) in PBS for 30 min. Then the cells were washed three times with PBS and observed under the microscope. 10 μM GF was added into the dish at 50 sec. The intracellular Ca²⁺ mobilization was monitored using a fluorescence microscope (IX83 system; Olympus, Tokyo, Japan) equipped with a band-path filter set (FITC; Olympus). The emission signal was recorded with a CCD camera. The fluorescent signals were recorded and analyzed using Olympus-analyzer software. The time courses of the fluorescence level of particular cells are expressed as the change in the fluorescent intensity normalized to the baseline-level fluorescence.