1. Vaginal Challenge:
A. Mechanical disruption:
Depo-Provera comes in a vial – 150mg/ml. Shake the vial well then dilute 1:5 in sterile PBS to a final concentration of 30mg/ml.
Inject each mouse with Depo-Provera 3mg in 100µl subcutaneously. Shake the diluted Depo-Provera well before drawing up the aliquot for each injection (see Critical Steps).
Make a 3% preparation of CMC by dissolving the powdered form in deionized water.
Make a 4% preparation of N-9 in 3%CMC. This step can be skipped if Conceptrol is going to be used. Tip – dilute the pure N-9 to 40% in water before working with it, and use positive displacement pipettes for making these preparations; they are too viscous for normal pipettes.
Prepare PsV inoculum in siliconized micro-tube by diluting purified PsV prep 1:4 in 3%CMC. The inoculum for each mouse consists of 5ul of PsV prep plus 15µl of 3%CMC (20µl total). For example, the preparation for 5 mice would be 30µl of PsV’s mixed with 90µl of 3%CMC (make an extra aliquot to account for sticking to the side of the tube, etc.). Mix well using the positive-displacement pipette until schlieren lines disappear.
After induction of anesthesia, place the mouse on its back and secure the tail by taping it to the operating surface.
Use the positive-displacement pipette to deposit 10µl of the inoculum into the mouse vaginal vault.
Advance the cytobrush into the vagina until resistance is encountered. Stabilize the genital tissues by gently grasping the anterior vaginal introitus (around the urethral opening) with smooth forceps. Twirl the cytobrush clockwise then counterclockwise 10 times.
Deposit the remaining 10µl of inoculum into the vaginal vault.
Occlude the introitus by gently grasping the vulva with forceps, then force the inoculum into the upper portion of the vagina and into the fornices by pushing the forceps cephalad under the pubic arch.
Euthanize mouse by CO2 inhalation.
Dissect out the reproductive tract.
For whole organ, multispectral imaging:
Incise the ventral vaginal wall sagitally. This opens the tubular structure of the vagina so it can be laid flat with the mucosa facing up.
Place the reproductive tract in PBS on ice. Imaging can be done up to 6 hours later.
Acquire and analyze multispectral images. For details, see the methods section of the paper.
For histological analyis:
Fill the vaginal vault with OCT freezing media. (Pipetting the OCT in with a 1000µl pipette tip works best).
Place the reproductive tract dorsal side down in the plastic freezing mold and fill the mold bed with OCT.
Snap freeze by placing the bottom side of the mold in contact with the surface of liquid nitrogen; be careful not to submerge the sample.
Cut 5-7µm sections on a cryotome and collect on slides. Fix by placing the slide in 2% paraformaldehyde in PBS for at least 10 minutes.
Mount with DAPI mounting media.
B. Chemical disruption:
Perform steps 1-6 above.
Draw up 50µl of the N-9 containing material (Conceptrol or 4%N-9 in 3%CMC) into the positive displacement pipette.
Advance the pipette tip into the vaginal vault. Gently grasp the vulva on either side of the pipette tip with smooth dissecting forceps to force retention of the N-9. Instill N-9.
Repeat step 10 from above.
Allow 6 hours to elapse.
Repeat step 6 from above.
Draw up 20µl of the previously prepared PsV inoculum into the positive displacement pipette.
Advance the pipette tip into the vaginal vault. Gently grasp the vulva on either side of the pipette tip with smooth dissecting forceps to force retention of the PsV inoculum. Instill the entire 20µl PsV inoculum.
Repeat step 10 from above.
Repeat steps 11-13 from above.
2. Endocervical Challenge
Conceptually, this is similar to the chemical disruption protocol described above. The main differences are:
a. Depo-Provera does not have any effect on the results.
b. A less viscous preparation of N-9 is used.
c. All the materials are pipetted directly into the cervical canal instead of into the vaginal vault.
- Make a 1% preparation of CMC by dissolving the powdered form in deionized water.
- Make a 4% preparation of N-9 in 1%CMC.
- Prepare PsV inoculum in a siliconized micro-tube by diluting the purified PsV prep 1:1 in 1%CMC. The inoculum for each mouse consists of 7µl of PsV plus 7ul of 1%CMC – 14µl total. For example, the preparation for five mice would be 42µl of PsV mixed with 42µl of 1%CMC (make an extra aliquot to account for sticking to the side of the tube, etc.). Mix well using the positive-displacement pipette until schlieren lines disappear.
- After induction of anesthesia, place the mouse on its back and secure the tail by taping it to the operating surface.
- Load 15µl of the 4%N-9 in 1%CMC preparation into a gel loading pipette tip.
- Place the mouse in 30° of Trendelenburg (head lower than feet). Ensure that there is adequate light. Insert closed dissecting forceps into the vagina and release them to gently dilate the introitus. Remove the forceps; insert the pediatric nasal speculum into the vagina to the level of the cervix and open the speculum, enabling direct visualization of the cervix. The cervical os, or opening, is usually anterior in the visual field (at 12 o’clock). Canulate the cervical os with the gel-loading pipette tip and advance the tip about 5-10mm into the cervical canal. Deposit the N-9 preparation into the cervical canal.
- Allow 6 hours to elapse.
- Repeat steps 4-6, depositing the 14µl aliquot of PsV inoculum into the cervical canal instead of the N-9 preparation.
- Follow the steps for histological analysis described in step #13 under Vaginal Challenge above. Take sections from several intervals, because the squamocolumnar junction, which is situated in the canal after it has split into two separate lumens, is easy to miss.