- Methylcellulose (MC): 25 centipoises. 2 w/v% MC stock
solution was prepared and stored as described in [4]. - Methylcellulose–sucrose: Mix 2 w/v % methylcellulose with
2.3 M sucrose 1:1 ratio and stir for at least 15 min at 4C
before use. Prepare fresh. - Roughened aluminum rivets.
- Ultracryomicrotome (e.g., Leica Microsystems Ultracut FCS)
to cut ultrathin frozen sections. - Diamond knife (e.g., Drukker International).
- Nickel grids: hexagonal, 50 mesh (VECO-Stork) supporting
a carbon-coated formvar film as described in [4](seeNote 4).
2.3 Probe
Preparation
- 1μL of DNA plasmid (pGEM-T including promoter SP6 and
T7 or Blue Script including promoter T7 and T3) can be used
to generate sense and antisense RNA probe using either
DIG-RNA labeling kit (e.g., Roche, cat. no. 11175025910)
or Biotin-UTP (e.g., Roche, cat. no. 11388908910). The
probes can be stored at 20 C.
2.4 ISH on Frozen
Sections
- 20SSC: 3 M NaCl, 300 mM sodium citrate.
- Prehybridization buffer: 50% deionized formamide (from a
minimum 99.5% stock), 2SSC from a 20stock in dH 2 O,
pH 6.5 treated with diethyl pyrocarbonate (DEPC). The buffer
must be freshly prepared. - Hybridization buffer: 50% deionized formamide, 2SSC (as
above), 10 w/v % dextran sulfate (sodium salt fromLeuconostoc
spp.), 50μg/mL heparin (sodium salt, grade I-A from porcine
intestinal mucosa, and 100μg/mLE. colitransfer RNA (from
Escherichia coliBacteria strain W) in DEPC-treated dH 2 O,
pH 6.5. - 2μg/mL DIG or biotin labeled antisense probe diluted in
hybridization buffer. - Hybridization chamber made of 6 and 9 cm glass petri dish-
es and filter paper soaked with 50% formamide in dH 2 O(see
Note 5and also [5]). - Hot plate at 55C.
- Microfuge caps.
2.5 Antibodies
and Detection
Antibodies and Protein-A Gold (PAG) conjugates are diluted in 1%
BSA in PBS. All dilutions should be prepared fresh.- 0.15 w/v % glycine in PBS.
- 1 and 0.1 w/v % BSA in PBS.
180 Catherine Rabouille