Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The overall goal of this procedure is to prepare epidermal sheets, which are suitable for herpes simplex virus infection studies. This is accomplished by first taking mirroring skin samples from adult tails or alternatively from newborn backs. Next, the epidermis is separated from the dermis by dys paste two treatment, then the epidermal sheets are floated on virus suspension.

Finally, the infected epidermal sheets are immunostain to detect viral infection and intermediate filaments. Ultimately, immunofluorescence microscopy is used to visualize the number of infected cells in the basal layer. This method can help answer key questions in the virology field, such as how herpes simplex virus invades epidermal tissue to reach its receptors and initiate infection.

Demonstrating the procedure will be narran, A postdoc and Katarina tier, a grad student from a laboratory. The preparation of epidermal sheets from sacrificed animals is carried out in strict accordance with the recommendations of the guide of Landes, amp, VE and CHU North RH West Flia. The study was approved by L-A-N-U-V and RW to begin prepare DMM containing high glucose and glutamine dipeptide with 10%FCS 100 international units per milliliter of penicillin and 100 micrograms per milliliter of streptomycin.

For the preparation of epidermal sheets, carefully dissolve dis space two powder to five milligrams per milliliter in heated PBS. Then pass the solution through a point 22 micrometer filter. The filter will be used directly for treatment For epidermal hole mounts.

Prepare blocking buffer with 0.5%milk powder point 25%gelatin from cold water, fish skin, and 0.5%Triton X 100. To prepare epidermis from newborn back skin after isolating the torso from a one to three day old mouse. According to the text protocol, use curved fine forceps to hold the torso in place and move blunt tip scissors underneath the skin in a cranial to coddle direction along the back to separate the skin from the torso.

Then slit the skin along the back with a scalpel. Chop the skin into one to two pieces, approximately 10 by 10 millimeters. Transfer the pieces into one well of a six well plate with approximately one milliliter of PBS, making sure the dermal side faces the bottom of the dish.

Remove the PBS and add 1.5 milliliters of disc based solution. Then incubate at 37 degrees Celsius for 30 minutes or four degrees Celsius overnight. After the incubation, use PBS to wash the tissue samples three times.

Next, use forceps to hold the dermis and a second set to gently remove the epidermis as an intact sheet. Then with its basal side facing down, transfer the epidermal sheet into DMM and immediately use the tissue for infection experiments. Alternatively, prepare epidermis from adult tail skin after sacrificing one to three month old mice and cutting off the tail according to the text protocol, use a scalpel to slit the tail lengthwise, peel off the skin from the bone and use a scalpel to chop it into five by five millimeter pieces.

Place up to four pieces in one well and proceed with dyse. Two incubation as just demonstrated to dissociate the epidermal cells. Place newborn epidermal sheets in a six well plate with the basal side floating on 1.5 milliliters per well of recombinant tripsin based cell dissociation solution.

Incubate at room temperature for 15 minutes, followed by 37 degrees Celsius for 15 minutes. After the incubation, add three milliliters of DM EM to stop the digestion and use curved fine forceps to dissociate the keratinocytes by gently moving the epidermis around. In the six well plate, collect the cell suspension and add an additional 1.5 milliliters of DMM to the wells.

Gently move the sheet of tissue before collecting the keratinocytes in medium. Again, repeat two more times. Alternatively, incubate the epidermal sheets on enzyme free CDS at room temperature for 15 minutes, followed by 37 degrees Celsius for 15 minutes.

After a second room, temperature 15 minute incubation. Gently pipette the CDS to flush out the keratinous sites. Collect the cell suspension and use fresh DM EM to repeat the flushing.

Three more times to perform HSV one wild type infection of an epidermal sheet. In one well of a 24 well plate Calculate the virus titer based on the estimated number of cells in the basal layer per epidermal sheets as shown here. Prepare a solution of HSV one in no less than 500 microliters of DM EM.Then remove the medium from the epidermal sheet and add approximately 100 platforming units per cell of HSV one in DM EM.Incubate at 37 degrees Celsius for one hour before replacing the viral medium with dmm.

To prepare epidermal hole mounts after various times of infection, use 3.4%formaldehyde to fix epidermal sheets at room temperature for two hours or at four degrees celsius overnight. Then after using PBS to wash the samples two times, add blocking buffer and incubate that room temperature for one hour. Replace the blocking buffer with a one to 60 dilution of mouse anti ICP zero and 100 nanograms per milliliter of rabbit polyclonal anti keratin 14 in blocking buffer to visualize viral gene expression and the intermediate filament network respectively.

Incubate at room temperature overnight the following day with PBS 0.2%Tween 20. Wash the tissue four to five times at room temperature over a period of four hours. Then add secondary antibodies as listed here and incubate at room temperature overnight.

Wash at room temperature for four hours as before. Then mount the epidermal sheets with their AAL side facing a specimen slide. Add fluorescent mounting medium and cover with a cover slip.

To prepare epidermal cryo sections, use 4%formaldehyde to fix epidermal sheets at room temperature for 20 minutes after using PBS to wash the slides two times, embed the fixed sheets in tissue freezing medium and freeze in liquid nitrogen. Then with a cryo microtome cut eight to 10 micrometer sections with 0.5%formaldehyde in PBS. Fix the sections again at room temperature for 10 minutes.

Then use PBS to wash the samples two times and block with 5%normal goat serum and 0.2%tween 20 in PBS for 30 minutes. At room temperature, use mouse anti ICP zero monoclonal antibody to stain the tissue for 60 minutes before washing three times with blocking buffer. Add secondary antibodies in blocking buffer listed here and incubate at room temperature for 45 minutes before washing three times with PBS.

Finally, add fluorescent mounting medium and cover with cover slips. The challenge of the method is to prepare epidermal sheets into which HSV one can penetrate from the basal layer after dispa two treatment to separate the epidermis from the dermis staining of the intermediate filament protein keratin 14 should ideally reveal an intact layer of epidermal basal cells while epidermal sheets from C 57 black six mice were smoothly separated by treatment with five milligrams per milliliter of dys space. Two, the same procedure disrupts the basal layer of the epidermis from B six A two G MX one mice as there are nearly no keratin 14 stained cells.

In this latter case, a lower concentration was necessary to preserve an intact basal layer as shown here. So far, no difference in efficiency of hsv. One infection has been seen between epidermis taken from the tails of adult mice or the backs of newborn mice, both the demonstrate efficient infection of the inter follicular epidermis as shown by the green staining of viral ICP zero HSV one infection depends on cholesterol as shown here by drug treatment depleting cholesterol from the plasma membrane infection of nin one deficient epidermis shows that nin one acts as a major receptor while HVEM plays a minor role.

After watching this video, you should have a good understanding of how to perform ex vivo infection of epidermic sheets to study invasion of herpes simplex virus into tissue. This tool offers the possibility to study cell determinants that play a role during the initial steps of viral infection in vivo.