All washes/rinses were performed in cyclindrical glass slide incubators (30mL):
All SSC washes were pre-heated to appropriate temps before proceeding
Hybridization solution was discarded and slides rinsed for ~30mins in 2x SSC.
Cover slips were removed.
Slides were washed twice in 2x SSC 15mins @ 40C.
Slides were washed three times in 1x SSC 15mins @ 40C.
Slides were washed once in 0.5x SSC 15mins @ 40C.
Tissue was equilibrated in Buffer 1 (100mM Tris-HCl, 10mM NaCl, pH = 7.5) 10mins @ RT.
Tissues were blocked with Blocking Buffer (Buffer 1 + 2% sheep serum + 0.3% Triton-X 100) for 1hr @ RT (500uL on each slide w/cover slips – sitting flat on bench top).
Antibody solution (Anti-Digoxigenin-AP Fab fragments [Roche - Exp Nov. 2010]) – Diluted anti-DIG 1:1000 in Blocking Buffer. Anti-DIG is stored @ 4C in FSH 240 on Lisa’s shelf.
Added 1mL of antibody solution to each slide and incubated without a cover slip for 2hrs @ RT – sitting flat on bench top.
Rinsed slides with Buffer 1 for 10mins, two times.
Rinsed slides with Buffer 2 (100mM tris-HCl, 100mM NaCl, 50mM MgCl2; pH = 9.5) for 10mins.
Incubated slides in substrate solution (30mL Buffer 2 + 135uL NBT [Roch - Exp Feb 2010 - 4-nitro blue tetrazolium] + 75uL BCIP [Roche Exp Nov 2010 - 5-Bromo-4-chloro-3-indolyl phosphate]) O/N @ RT in the dark. Both NBT & BCIP are stored @ -20C in FSH 236 in the “ISH Reagents” box.
To test out the viability of these RLOv ISH probes (from 20151109) and not waste black abalone slides if this doesn’t work, I selected three unstained red abalone post-esophagus sections:
RLO – NO PHAGE
RLO STIPPLED – NO PHAGE
All slides were processed in a single, horizontal glass slide incubator (200mL), unless otherwise noted.
All steps were conducted at room temperature (RT), unless otherwise noted.
DEPARAFFINIZATION & REHYDRATION
All slides were deparaffinized with three changes of xylene (SafeClear II; Fisher) for 10mins each.
Slides were hydrated with a graded ethanol series (100%, 100%, 80%, 70%, 50%) for 3mins each.
Slides were rinsed with molecular grade H2O.
Tissue sections were equilibrated in Tris Buffer (0.2M Tris-HCl, 2.0mM CaCl, pH = 7.2) for 5mins.
Tissues were permeabilized for 1.5hrs in preheated 50ug/mL Proteinase K (Qiagen) in Tris Buffer @ 56C.
Slides were rinsed with 1x PBS three times, 10mins each.
Slides were incubated 30mins in 30mL Prehybridization Buffer (50% deionized formamide, 4x SSC) @ 53C in a cylindrical glass slide incubator due to limited volume of deionized formamide available:
Prepared probes by boiling 3mins and immediately incubating in ice water bath for 30mins.
Slides were rinsed with 2x SSC and air dried for 5mins.
Probes were diluted 1:300 in 1000uL of Prehybridization Buffer. All three negative control probes (indicated by “-C” in subsequent labeling) were combined into a single dilution.
NOTE: RLOv Membrane Gene 2 probe was ruined because boiling water got into the tube during denaturation. This didn’t happen to any of the other tubes that were all boiled at the same time. Not sure what happened. However, this may have worked out OK because I did not pull enough slides to accomodate the negative control probes. So, now that I’m not able to test three probes, I can use the negative control probes!
300uL of probe solutions and cover slip were added to the following slides:
The three groups of slides were placed into separate slide cases and a 1mL of Prehybridization Buffer was added to each case (to maintain high humidity during incubation).
The cases were incubated on their sides O/N @ 53C.