Immunofluorescent Detection of Viral Infection in Murine Epidermal Whole Mounts

Samenvatting

Source: Rahn, E., et al. Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1. J. Vis. Exp. (2015)

This video demonstrates the immunofluorescent detection of herpes simplex virus type 1 infection in murine epidermal whole mounts, enabling visualization of viral protein expression and its distribution within basal keratinocytes by confocal microscopy.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 

1. Infection of Epidermal Sheets with HSV-1(Herpes simplex virus type 1)

1. Prepare a solution of HSV-1 wt (wild-type) strain 17 in not less than 500 µl DMEM (Dulbecco’s modified Eagle medium) and replace the medium with the virus solution in which the epidermal sheets are floating. This time point is defined as time 0. Perform infection of an epidermal sheet in one well of a 24-well plate at 37 °C. The calculation of the virus titer is based on the estimated number of cells in the basal layer per epidermal sheet (approximately 2 x 10⁵ cells per 5 x 5 mm sheet).
2. Infect with HSV-1 at approximately 100 plaque-forming units (PFU)/cell and replace the virus-containing medium with DMEM at 1 hr post-infection. 
   NOTE: In case of epidermal sheets from newborn skin, keep in mind that sheets start to dissociate during incubation.

2. Visualization of Infected Cells

1. Epidermal Whole Mounts
   1. Fix epidermal sheets with 3.4% formaldehyde either for 2 hr at RT or O/N at 4 °C. Wash two times with phosphate-buffered saline (PBS) and block with 0.5% milk powder, 0.25% gelatin from cold water fish skin and 0.5% Triton X-100 in Tris-buffered saline (TBS) for 1 hr at RT.
   2. Incubate the sheets O/N with mouse anti-ICP0 (Infected-cell protein 0, monoclonal antibody 11060) diluted 1:60 in blocking buffer at room temperature (RT). To visualize the intermediate filament network, incubate simultaneously with rabbit polyclonal anti-mouse keratin 14 (100 ng/ml).
   3. Wash 4 to 5 times with PBS-0.2% Tween-20 for 4 hr at RT. Incubate O/N with AF488-conjugated anti-mouse IgG (1 µg/ml), AF555-conjugated anti-rabbit IgG (1 µg/ml), and DAPI (4’, 6-diamidino-2-phenylindole) (100 ng/ml) in blocking buffer at RT.
   4. Wash with PBS-0.2% Tween 20 for 4 hr at RT. Mount epidermal sheets with their basal side on top of a specimen slide, embed in fluorescent mounting medium and cover with coverslips.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
DMEM/high glucose/GlutaMAX	Life Technologies	31966047	Needed for cultivation of epidermal sheets
Dispase II powder	Roche	4942078001	Has to be solved in heated PBS
Enzyme-free cell dissociation solution	Sigma	C5914	Needed for very gentle dissociation of epidermal sheets
TrypLE select cell dissociation solution	Life Technologies	12563-029	Needed for dissociation of epidermal sheets
Chelex 100 resin	Bio-Rad	142-2832	Needed for chelation of polyvalent metal ions from the fetal calf serum
Gelatin from cold water fish skin	Sigma	G7765	Needed for minimization of non-specific antibody binding
Keratin 14 Polyclonal antibody (AF64) (conc.: 1 mg/ml)	Covance	PRB-155P	Used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis
Mouse IgG (H+L) Secondary antibody, Alexa Fluor 488 conjugate (conc.: 2 mg/ml)	Life Technologies	A-11029	Used as secondary antibodies
Rabbit IgG (H+L) Secondary antibody, Alexa Fluor 555 conjugate (conc.: 2 mg/ml)	Life Technologies	A-21429	Used as secondary antibodies
4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride) (conc.: 0.1 mg/ml)	Sigma	36670	Used to counterstain the nucleus