#### ^Indirect\ Immunofluorescence^

#### ^Equipment\ and\ reagents^

· Glass coverslips, 22x22mm

· ^6-well\ plates^

· ^Paraformaldehyde\ (granular;\ Electron\ Microscopy\ Sciences)^

· ^Parafilm^

· ^Forceps,\ Dumont,\ GG\ (Electron\ Microscopy\ Sciences)^

· ^Fluorescently\ labeled\ secondary\ antibody\ (Vector\ Laboratories)^

· ^Mounting\ medium\ (Molecular\ Probes)^

· ^Microscopy\ coverslide^

#### ^Method^

1\. Prepare sterile 22x22mm No. 1.5 glass coverslips by soaking them in
95% ethanol^a^ and gently flaming them. For multiple samples use 6-well
plates^b^

2\. ^Grow\ cells\ on\ coverslips\ to\ 50-70%\ confluencec^

3\.
^Fix\ cells\ in\ 3-4\ ml\ 2%\ formaldehyde\ in\ PBS,\ pH\ 7.4\ for\ 15\ min.\ at\ room\ temperatured,e^

4\.
^Quickly\ rinse\ cells\ in\ 4\ ml\ PBS,\ pH\ 7.4\ and\ wash\ the\ coverslips\ in\ 2\ ml\ PBS,\ pH\ 7.4,\ 2\ X\ 5\ min.\ at\ room\ temperaturef^

5\.
^Permeabilize\ cells\ by\ removing\ the\ wash\ solution\ and\ adding\ 2\ ml\ of\ 0.5%\ Triton\ X-100\ in\ PBS,\ 7.4\ for\ 5\ minutes\ on\ ice.^

6\.
^Quickly\ rinse\ cells\ in\ 4\ ml\ PBS,\ pH\ 7.4\ and\ wash\ the\ coverslips\ in\ 2\ ml\ PBS,\ pH\ 7.4,\ 3\ X\ 8\ min.\ at\ room\ temperature.^

7\.
^Place\ a\ piece\ of\ Parafilm\ large\ enough\ to\ easily\ accommodate\ all\ coverslips\ on\ a\ flat\ surface.^

8\.
^For\ each\ coverslip,\ place\ 50\ ul\ of\ the\ appropriate\ concentration\ of\ primary\ antibody\ on\ the\ Parafilm.\ Make\ sure\ to\ separate\ the\ drops\ generously.^

9\.
^Remove\ the\ coverslip\ from\ the\ 6-well\ plate\ using\ fine\ forceptsg.\ Remove\ excessive\ PBS\ by\ blotting\ the\ edges\ of\ the\ coverslip\ gently\ against\ a\ piece\ of\ filter\ paper.^

10\.
^Invert\ the\ coverslip\ with\ the\ cells\ facing\ down\ onto\ the\ antibody\ drop.^

11\.
^Incubate\ the\ coverslips\ for\ 1h\ at\ room\ temperature\ inverted\ onto\ a\ piece\ of\ Parafilm.\ Cover\ the\ Parafilm\ with\ a\ light-tight\ lid.^

12\.
^Remove\ the\ coverslip\ from\ the\ Parafilm\ by\ squirting\ 1\ ml\ of\ PBS,\ pH.\ 7.4\ under\ the\ coverslip\ to\ float\ it\ up.\ Place\ the\ coverslip\ with\ the\ cells\ facing\ up\ in\ the\ 6-well\ plate.^

13\.
^Wash\ the\ coverslip\ in\ 2\ ml\ PBS,\ pH\ 7.4,\ 3\ X\ 8\ min.\ at\ room\ temperature.^

14\.
^Repeat\ steps\ 6-9\ with\ a\ drop\ of\ appropriately\ diluted\ secondary\ antibody\ (2-4\ ug/ml\ in\ PBS,\ pH\ 7.4).^

15\.
^Incubate\ the\ coverslip\ for\ 1\ h\ at\ room\ temperature\ inverted\ onto\ a\ piece\ of\ ^

16\.
^Wash\ the\ coverslip\ in\ 2\ ml\ PBS,\ pH\ 7.4,\ 3\ X\ 8\ min.\ at\ room\ temperatureh^

17\.
^Place\ 10\ ul\ of\ mounting\ medium\ on\ a\ glass\ microscopy\ slide.\ Remove\ the\ coverslip\ from\ the\ 6-well\ plate.\ Remove\ excessive\ PBS\ by\ blotting\ the\ edges\ of\ the\ coverslip\ gently\ against\ a\ piece\ of\ filter\ paper.^

18\.
^Invert\ the\ coverslip\ cells\ facing\ down\ onto\ the\ mounting\ mediumi.^

19\.
^Remove\ excessive\ mounting\ medium\ by\ holding\ two\ pieces\ of\ filterpaper\ against\ two\ opposing\ edges\ of\ the\ coverslipj.^

^aIt\ is\ most\ convenient\ to\ store\ a\ large\ number\ of\ coverslips\ in\ 95%\ ethanol.^

^bAll\ volumes\ given\ are\ optimized\ for\ 6-well\ plates\ and\ 22x22\ mm\ coverslips.^

^cConfluent\ lawns\ of\ cells\ result\ in\ increased\ background\ signal.^

^d2%\ formaldehyde\ is\ made\ up\ fresh\ prior\ to\ use\ by\ dissolving\ the\ appropriate\ amount\ of\ EM\ grade\ paraformaldehyde\ in\ PBS\ in\ a\ Pyrex\ bottle\ on\ a\ hot\ plate\ with\ a\ stir\ bar.\ Paraformaldehyde\ goes\ into\ solution\ at\ 700C.\ Do\ not\ tighten\ the\ bottle\ cap\ so\ as\ to\ avoid\ build-up\ of\ pressure.\ Place\ the\ dissolved\ solution\ on\ ice\ to\ cool\ down\ and\ check\ pH.^

^eAlternative\ fixation\ procedures\ include\ methanol\ at\ -20C\ for\ 5\ min\ or\ 3.7%\ paraformaldehyde,\ 5%\ acetic\ acid\ for\ 20\ min\ (4).\ Since\ both\ of\ these\ methods\ also\ permeabilize\ membranes,\ skip\ to\ step\ 5.^

^fIt\ is\ most\ convenient\ to\ use\ a\ vacuum\ suction\ device\ to\ remove\ the\ solution\ from\ the\ 6-well\ plate\ and\ to\ simultaneously\ add\ the\ new\ solution\ using\ a\ 10ml\ pipette.\ It\ is\ crucial\ that\ the\ cells\ on\ the\ coverslip\ never\ dry\ out.^

^gWe\ recommend\ Dumont\ GG\ forcepts\ (Electron\ Micrsoocpy\ Sicences).^

^hNuclei\ can\ be\ counterstained\ for\ easy\ visualization\ by\ use\ of\ DAPI\ or\ Hoechst\ 33342.\ After\ two\ washes\ as\ described\ in\ step\ 14,\ incubate\ coverslips\ for\ 5\ min\ in\ 2\ ml\ 500\ ng/ml\ DAPI\ or\ Hoechst\ 33342\ and\ wash\ twice\ more\ as\ described\ in\ step\ 14.\ DAPI\ and\ Hoechst\ give\ a\ blue\ stain.^

^iVectashield\ (Vector\ Laboratories)\ or\ mouting\ medium\ from\ Molecular\ Probes\ is\ recommended.^

^jA\ good\ indication\ that\ all\ excessive\ mounting\ medium\ has\ been\ removed\ is\ if\ the\ coverslip\ can\ not\ be\ moved\ on\ the\ coverslide\ when\ gently\ pushed\ with\ the\ filterpaper.^

·
^Suspension\ cells\ can\ either\ be\ centrifuged\ onto\ a\ coverslip\ in\ a\ Cytospin\ centrifuge\ and\ then\ processed\ as\ described^*^.^*^\ Alternatively,\ cells\ can\ be\ labeled\ in\ suspension\ as\ described\ above^*^.^*^\ Start\ out\ with\ about\ 3\ x106\ cells\ and\ at\ each\ step,\ spin\ the\ cells\ for\ 2\ min\ at\ 3\ 000\ xg\ and\ carefully\ remove\ the\ supernatant.\ Antibody\ incubations\ are\ done\ in\ a\ volume\ of\ 100ul\ on\ a\ rocking\ platform.\ After\ the\ last\ wash\ re-suspend\ the\ cells\ directly\ in\ 50\ ul\ mounting\ medium\ and\ spot\ 15\ ul\ on\ a\ microscopy\ slide\ and\ cover\ with\ an\ empty\ coverslip.^

·
^This\ protocol\ can\ be\ used\ for\ detection\ of\ multiple\ proteins.\ Generally\ results\ are\ not\ compromised\ by\ the\ simultaneous\ incubation\ of\ multiple\ primary\ or\ secondary\ antibodies.\ If\ detection\ of\ mutiple\ proteins\ is\ done\ step-wise\ it\ is\ recommended\ that\ steps\ 7-16\ are\ carried\ out\ for\ the\ first\ antibody\ and\ then\ repeated\ for\ the\ second\ antibody.\ In\ either\ case\ it\ is\ essential\ to\ ensure\ that\ the\ primary\ antibodies\ used\ are\ from\ different\ species\ (polyclonal\ from\ rabbit\ and\ monoclonal\ from\ mouse\ are\ commonly\ used)\ and\ that\ the\ secondary\ antibodies\ do\ not\ cross-react\ between\ these\ species.\ When\ doing\ multiple\ labels\ the\ recommended\ sequence\ of\ fluorophores\ is\ fluorescin,\ Texas\ Red,\ Cye\ 5,\ AMCA.\ Note\ that\ Cye\ 5\ emits\ in\ the\ infrared\ part\ of\ the\ spectrum\ and\ requires\ the\ use\ of\ an\ appropriate\ cooled\ CCD\ camera\ for\ detection.\ Indirect\ immunofluroescence\ can\ be\ combined\ with\ in\ situ\ hybridization\ methods\ to\ detect\ RNA\ or\ DNA.^