A. The Pan-Polyomavirus Immunohistochemistry Test (P-PIT) makes use of a cocktail of three antibodies which in combination detects T antigens from all human polyomaviruses. 293HEK cell pellets expressing human polyomavirus T antigens.
B. P-PIT recognizes infected cells in human diseases associated with polyomaviruses: JCV infected oligodendrocytes in Progressive Multifocal Leukoencephalopathy; MCV positive Merkel cell carcinoma; HPyV7 infection in pruritic epidermal hyperplasia syndrome in patients following lung transplant; and TSV infected inner hair shaft cells in Trichodysplasia Spinulosum. This makes screening archival FFPE cases easy and can be applied to tissue microarrays to rapidly assess hundreds of cases simultaneously.
P-PIT Immunohistochemistry Staining Protocol
1) Bake slides @ 60°C (preheat the oven) — 60 min
2) Deparaffinize slides:
a) Incubate the slides in xylene, agitate occasionally — 10 min
b) Remove the slides from the first xylene-containing dish and immediately place in the second xylene-containing dish. Incubate the slides in xylene, agitate occasionally — 10 min
3) Rehydrate slides through graded alcohol to milliQ water:
a) 100% ethanol x 2 10 min each
b) 95% ethanol x 2 5 min each
c) 80% ethanol x 1 50 dips
d) 70% ethanol x 1 50 dips
e) Place in milli-Q water 2-3 changes (fresh each time)
4) Set up incubation area: (when completely finished, discard towels and let air dry):
a) Wet paper towels and set into empty slide box (humidified chamber)
b) Never let tissue dry out, handle one slide at a time
5) Quench endogenous peroxidase:
a) Cover the tissue sections with 3% hydrogen peroxide (freshly diluted from 30% stock solution) — 10 min
b) Place the slides a plastic coplin jars
c) Rinse with several changes of milli-Q water
6) Perform heat induced epitope retrieval:
a) Fill plastic jars with 1mM EDTA buffer pH 8.0 and close the plastic lid
b) Add 500 mL milliQ water to chamber, place in jars with slides, close lid (put the last slide facing inside)
c) Decloaking Program (BioMedical Chamber):
i) Setting 1: 125oC for 3:15 min (must press “start/stop” to continue) ~15 min (varies!)
ii) Setting 2: 90oC for 0:15 min (must press “start/stop” to end) ~19 min (varies!)
d) Let cool the copin jar on bench top — 60 min
e) Rinse the slides with milliQ water x 2 — 5 min each
f) Transfer slides to glass coplin jars and rinse with 1 x TBS buffer 5 min
7) Blocking: (Protein block DAKO, protein-free)
a) Blot off excess liquid with pieces of Wattman paper
b) Circle the tissue with hydrophobic pen
c) Add about 100ul for blocking buffer (adjust for large / small samples) — 10 min
8) Antibody Incubations, Staining, & LOTS of Washes:
a) Quickly remove excess liquid from the slides, add primary antibody diluted in antibody diluent (see Solutions) and incubate in a humidified chamber —1h RT
pAB416 (Millipore DP-02) 1:200
XT7 1:50 (concentrated hybridoma supernatant)
2T2 1:100 concentrated hybridoma supernatant)
i) Rinse the slides with TBS once briefly and then x 3 with agitation — 5 min each
b) Quickly remove excess liquid from the slides and cover the tissue with ~2-4 drops of secondary antibody
(DAKO/BioCare). Incubate the slides in a humidified chamber — 30 min RT
i) Rinse slides with TBS buffer x 3 with agitation — 5 min each
c) Quickly remove excess liquid from the slides and cover the tissue with ~2-4 drops DAB Chromogen
(DAKO/BioCare). Incubate the slides — 5-10 min
i) Rinse slides with milli-Q water x 3, agitate well!
d) Counterstain slides with 1x Hematoxylin — 10-15 sec
Use a syringe with a filter add couple of drops to cover the slides
i) Rinse in tap water x 3 — 1 dip each
e) Dip in 1% lithium carbonate — 5 dips
i) Rinse in tap water x 3 — 1 dip each
f) Dehydrate slides
i) 95% ethanol x 2 — 30 dips
ii) 100% ethanol x 2 — 30 dips
g) Clear slides in xylene x 2 — 5 min each
9) Finalize slides (chemical/fume hood)
a) Wipe off excess liquid xylene with whatman paper
i) Wipe off bottom of slide
ii) Wipe off the top of the slide around the tissue
b) Set slides down 1 at a time on recycled whatman sheet, above bench
(avoid setting right on permount edge, get a new sheet if necessary)
c) Add ~1-2 drops permount or other xylene / toluene compatible mounting medium
d) Place large or small coverslip depending on tissue size
e) Press down firmly, try to avoid smearing permount
f) Wipe of edges and flip slide upside down
g) Let it dry at RT.
Solutions:
Antibody Diluent (100mL, keep at 4C)
1% BSA = 1g
0.1% gelatin = 0.1g
0.5% Triton X-100 = 0.5mL @ 100%
0.05% sodium azide = 0.05g
Bring to 100mL in PBS, pH 7.4
1x Tris-buffered Saline (TBS): 50 mM Tris-HCl, pH 7.5, 150 mM NaCl