Indirect immunofluorescence คือ

3.3.4 Fluorescent antibody test

Immunofluorescence assays (IFAs), ELISA, and immunoblot use antibodies to recognize characteristic pathogen in silkworm tissues and localization of infections (in situ) on fixed specimens, but needs to be examined by fluorescence microscopy. β-Tubulin antibodies were utilized for identifying different phases of N. bombycis by indirect IFA (Chen et al., 2017; Huang et al., 2018; Qian, Guo, & Hu, 1986).

Fix infected cells or tissue slices in 4% formaldehyde at room temperature for 10 min and permeabilized with PBS-Triton X-100 for 20 min (Dang et al., 2013)

Wash samples three times with PBS + 0.1% Triton X-100 (PBST) and block in PBST containing 10% goat serum and 5% BSA for 1 h at 37 °C

After three washes, incubate samples for 1 h at 37 °C with anti-Nosema antisera or IgG (rabbit) or anti-Nosema MAbs (mouse) diluted in blocking solution containing 2% Triton X-100 (1:200)

After washing with PBST, maintain the samples in dark for 1 h at 37 °C with Alexa Fluor 488 or 594 conjugate Goat anti-mouse IgM (MAbs) or anti-rabbit IgG (PAbs)

Wash samples in PBST and stain in DAPI (4′6-diamidino-2-phenylindole, Sigma) for 5 min for nucleus labelling

After washing with PBST, add ProLong1 Gold anti-fade reagent (Thermo Fisher) and observe cells using an Olympus FV1200 laser scanning confocal microscope

Presence or absence of chitin in different phases of microsporidian life cycle allows easy labelling. β-Tubulin antibodies were utilized for the identification of different phases of N. bombycis by IFA.

Label N. bombycis by β-Tubulin antibodies for identification of different phases by an indirect immunofluorescence assay

Hepatitis A virus-specific BSC-1 cells were used for the detection of serum immunoglobulins to hepatitis A virus by indirect immunofluorescence. Of 150 serum samples tested, specific immunoglobulin M was detected only in patients with serologically confirmed acute hepatitis A, while specific immunoglobulin G was detected in patients with acute or past clinical hepatitis A as well as many patients with no known history of hepatitis.

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