Visualization of Viral Double-Stranded RNA and Host Pattern Recognition Receptors in Infected Epithelial Cells

الملخص

Source: Mateer, E., et al., Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection. J. Vis. Exp. (2019)

This video demonstrates the confocal imaging of viral double-stranded RNA (dsRNA) and cytoplasmic pattern recognition receptors (PRRs) in virus-infected epithelial cells. It highlights how antibody-based fluorescent labeling reveals the interaction between viral replication intermediates and host immune sensors.

البروتوكول

1. Fixation and immunostaining

1. Aspirate the media. Rinse the cells by adding 1 mL of phosphate-buffered saline (PBS) supplemented with calcium and magnesium to each well.
2. Remove PBS. Add 1 mL of methanol (MeOH) pre-chilled at -20 °C and incubate at -20 °C or on dry ice for 15 min.
3. Remove MeOH.
4. Add 1 mL of PBS to each well, and wash samples at 4 °C with gentle rocking for 5 min. Repeat the wash for a total of 4 times.
5. Wash the fixed cells on coverslips in 1 mL of 0.2% t-octylphenoxypolyethoxyethanol for 5 min at 4 °C, with gentle rocking.
6. Add 1 mL of PBS to each well. Wash samples at 4 °C for 5 min with gentle rocking. Repeat the washing step a total of 4 times.
7. To detect dsRNA and MDA5 (Melanoma differentiation-associated protein 5), incubate samples in 200 μL of primary antibodies diluted in 3% bovine serum albumin (BSA). Dilute the anti-dsRNA 9D5 antibody at a 1:2 dilution and dilute the anti-MDA-5 antibody at 1:250. Incubate with gentle rocking at 4 °C overnight.
8. Remove primary antibodies and wash each well with 1 mL of PBS for 5 min with gentle rocking at room temperature (RT). Repeat this wash four more times.
9. Add 200 μL of secondary antibodies (1:2,000 dilution) diluted in 3% BSA and incubate at RT for 1 h.
10. Remove secondary antibodies and wash each well with 1 mL of PBS for 5 min with gentle rocking at RT. Repeat this wash four more times.
11. To detect JUNV nucleoprotein (NP) and RIG-I, add 200 μL of conjugated antibodies diluted in 3% BSA. Dilute the conjugated anti-JUNV NP (AG-12) at 1:1,000 and incubate samples for 2 h at RT with gentle rocking. Dilute the conjugated anti-RIG-I antibody at 1:500 and incubate at 4 °C overnight with gentle rocking.
12. Remove conjugated antibodies and wash each well with 1 mL of PBS for 5 min with gentle rocking at RT. Repeat this wash four more times.
13. Counterstain the coverslips with 4′,6-diamidino-2-phenylindole or DAPI (1:1,000) for 3 min with gentle rocking at RT.
14. Wash the coverslips 3 times, each time for 5 min in 1 mL of 0.5% t-octylphenoxypolyethoxyethanol with gentle rocking at RT.
15. Wash twice in 1 mL of PBS for 5 min each time with gentle rocking at RT.
16. Wash once in 1 mL of ddH₂O for 1 min at RT, rocking gently.
17. Mount coverslips onto glass slides using mounting media. Let it cure overnight.
18. Seal the slides with nail polish and air-dry for 1 h.
19. Image on confocal microscope with the 60x/1.42 numerical aperture oil immersion lens using the same laser emissions for each sample.
20. When analyzing the data, if necessary, make adjustments for brightness and contrast using the same linear adjustment for all samples.

المواد

قائمة المواد المستخدمة في هذه المقالة

الاسم	الشركة	رقم فهرسي	التعليقات
APEX Alexa Fluor 647 antibody labeling kit	Invitrogen	A10475
BSA (Bovine serum albumin)	Sigma Aldrich	A4503
DAPI (4′,6-diamidino-2-phenylindole.)	Cell Signaling	4083	1:1,000 dilution
Donkey-anti rabbit Alexa Fluor 594	Invitrogen	A-11058	1:2,000 dilution; Lot #: 1454437
Dulbecco's modified Eagle's medium	Corning	10-013-CV
Fetal Bovine Serum	Atlanta Bio	S11150
Glass microscope slides	Fisher	12-550-15
Goat-anti mouse Alexa Fluor 488	Invitrogen	A-11029	1:2000 dilution; Lot #: 1874804
Human lung epithelial A549 cells	ATCC	CCL-185
Methanol	Fisher	A412	Stored at -20 °C
Mouse MAb anti-JUNV NP	BEI	NA05-AG12	Conjugated to Alexa-647 at 1:1,000 dilution
Mouse MAb pan-Enterovirus 9D5 Reagent	Millipore Sigma	3361	ready for use; diluted to 1:2; Lot #: 3067445
PBS supplemented with Ca and Mg	Corning	21-030-CV
PDL Coated coverslips	Neuvitro	H-12-1.5-pdl
ProLong Gold antifade	Invitrogen	P10144
Rabbit MAb MDA-5	Abcam	ab126630	1:250 dilution; Lot #: GR97758-7
Recombiant Candid#1 strain of JUNV	Lab generated	Lab generated	As previously described in reference 13.
RIG-I mouse MAb conjugated to Alexa-594	Santa Cruz	sc-376845	1:1000 dilution; Lot #: AO218
Triton X-100	Sigma Aldrich	T8787