Immunostaining to Visualize Viral Antigen and Host Protein Colocalization in the Insect Gut

Аннотация

Source: Zhang, L., et al. Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts. J. Vis. Exp. (2021)

The video demonstrates the preparation of gut tissue from a plant virus–infected insect vector for fluorescence imaging. Fixed guts were washed, permeabilized, and incubated with two fluorescent antibodies in a blocking solution—one targeting a viral antigen in vesicles, the other a vesicle-associated membrane protein. The tissue was counterstained for actin filaments, mounted with a nuclear stain, and imaged to reveal in situ colocalization of viral and host proteins.

Протокол

1. Nonviruliferous insect rearing

1. Collect white-backed planthoppers (WBPHs) from rice fields and rear with rice seedlings in 1 L glass beakers covered with insect-proof net in an incubator at 28 °C with 16 h light and 8 h dark. Because southern rice black-streaked dwarf virus (SRBSDV) is not transmitted via eggs, newly hatched nymphs are not viruliferous.
2. With a brush pen, gently brush insects from the beaker, rearing insects into a new beaker of fresh rice seedlings each week until WBPH nymphs have hatched. Continue rearing these hatched nonviruliferous nymphs to 2- or 3-instar.
   NOTE: Brush carefully to avoid WBPHs flying fro....

Материалы

Список материалов, использованных в этой статье

Имя	Компания	Каталожный номер	Комментарии
3% Bull serum albumin (BSA)	Coolaber	SL1331	Dilute antibodies
Cover glass	Solarbio	YA0771-18*18mm	For slide making
Dissecting microscope	Beitja	XTL-7045B1	For insect dissection
Laser scanning confocal microscope	Zeiss	Zeiss LSM880	Observe fluorescence signal
Microscope slides	Solarbio	ZBP-7105	For slide making
Mounting medium with 4'6-diamidino-2-phenylindole (DAPI)	Abcam	AB104139	Label cell necleus
Paraformaldehyde	Sigma	158127	For tissues fixation
Phalloidin	Invitrogen	A22284	Label actin of midgut epithiels
Triton X-100	Amresco	0290C484	For tissues permeation
Tweezers (5-SA)	AsOne	6-7905-40	For insect dissection