3. Methods
3.5. Immunostaining and Whole-mount in situ hybridization (WISH)
Upon the MEMFA fixation the AC were washed 3 times with PBS containing 0.1% Tween20 (PTw) and stained according to the following protocol:
Blocking with 10% fetal calf serum (FCS, Difco) in PTw – 1h at room temperature Rinse with PTw – 1 time
Application of the primary antibodies (α-GFP, α-HA, α-pJNK, α-β1integrin), diluted in PTw – for 2-3h at room temperature or at 4°C overnight
Wash with PTw – 7 min, 3 times
Application of the secondary antibodies (α-myc-Cy3, α-rabbit-FIC, α-mouse-Alexa488, α-mouse-Cy5)
Wash with PTw – 7 min, 3 times
DAPI stain – optional: for the DAPI stain the AC should be rinsed with PBS and incubated in 1µg/ml DAPI PBS solution for 7-10 min, and washed 2 times with PBS.
Upon the immunostaining protein localization was analyzed by laser scanning microscopy on LSM 510Meta, Zeiss.
In addition, the immunostaining with α-pJNK antibodies the PTw should be substituted by TBST (20mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20), and the incubation with the primary antibodies should be done in PTw with 10% FCS
3.5.2 Immunostaining of the gelatin-albumin sections
Vibratome sectioning (Hollemann et al., 1999): embryos were embedded in gelatin-albumin (GA), which was polymerized by mixing 2 ml of GA with 125 µl of 25% glutaraldehyde. 50 µm vibratome sections were made using a Leica VT1000S Vibratome. Sectioned tissue samples were immunostained according to the same procedure as animal caps. GA and mowiol were made as follows: Gelatin/albumin: 4.88 mg/ml gelatin, 0.3 g/ml bovine serum albumin, 0.2 mg/ml sucrose in PBS. The gelatin was dissolved by heating the solution to 60°C. Albumin and sucrose were added, filtered with a 0.45 μm filter (Satorius) and stored at -20°C. Mowiol: 5 g Mowiol was stirred overnight in 20 ml PBS. After addition of 10 ml glycerol, the solution was stirred again overnight. Not dissolved Mowiol was collected by centrifugation for 30 min at 20,000 g. The supernatant was pH adjusted to pH~7.0 (using pH stripes) and stored at -20°C.
3.5.3 Whole mount in situ hybridization (WISH)
To determine the expression pattern of the selected genes, whole mount in situ hybridization was employed. By this method the gene specific RNA can be visualized by the hybridization with the labeled antisense probe, which could be recognized by specific antibody, coupled with alkaline phosphatase.
In the cases, when a change of gene expression needs to be observed, the injected side was labeled with ß-galactosidase and the X-gal staining was performed prior to the in situ.
Embryo fixation and X-gal staining
To be directly analyzed by WISH the embryos were fixed in 1x MEMFA solution for 1 h at room temperature and stored in 100% ethanol. In cases, when X-gal stain was required, the following protocol was used:
MEMFA fixation: 20 min at room temperature Wash with PBS: 3x, 10 min
Application of the X-gal staining solution: 20-30 min at room temperature Wash with PBS: 3x, 5 min
MEMFA fixation: 40 min at room temperature Store embryos in 100% ethanol
Rehydration of embryos
Prior to in situ embryos were rehydrated, as it is described in the Table 3.4
Table 3.4. Rehydration of embryos
Solution Incubation time
100% ethanol 5min
75% ethanol in water 5min 50% ethanol in water 5min 25% ethanol in PTw 5min
PTw 5min
Proteinase K treatment
To make embryos accessible for RNA probes, they were treated with proteinase K (10µg/ml) in PTw. And the proteinase K incubation time was chosen depending on the embryo stage (Table 3.5).
Table 3.5 Proteinase K treatment
Stage Incubation time
(min)
Temperature
9-10.5 6-8 RT
14-16 8-10 RT
20-25 15-18 RT
36 22-25 RT
40 17-20 37°C
42-43 27-30 37°C
46 32-35 37°C
Acetylation and refixation
Following the proteinase K treatment the embryos undergo acetylation (Table 3.6) and refixation.
Table 3.6 Acetylation
Buffer Incubation time
1M Triethanol amine HCl (TEA), pH 7.0 2x 5min 1M TEA with 0.3% acetic anhydride 5 min 1M TEA with 0.6% acetic anhydride 5 min
PTw 5 min
Upon the acetylation, embryos were fixed for 20 min in 4% formaldehyde in PTW and washed 5 times with PTw subsequently after the fixation
Hybridization
After the last washing step, 1ml PTw was left in the tubes and 250 µl Hyb-Mix were added. The solution was replaced immediately by 500 µl fresh Hyb-Mix and incubated for 10 minutes at 60°C. Hyb-Mix was exchanged again (1ml Hyb-Mix was added) and embryos were incubated 4-5h at 60°C. After incubation, the Hyb-Mix was replaced with the desired RNA probe, diluted in Hyb-Mix solution. The hybridization took place overnight at 60°C and mild shaking.
Washing
To remove unspecific bound RNA probes, the samples were washed and digested with RNAse A (10 µg/ml) and RNAse T1 (10 U/ml) (Table 3.7).
Table 3.7 Washing and RNAse treatment
Solution Incubation temperature (°C) Incubation time (min)
Hyb Mix 60 10
Prior to incubation with the Sheep AP-coupled anti-Dig antibody (sigma), embryos were incubated in blocking solution, containing MAB, BMB and horse serum to block unspecific binding sites according to the scheme, indicated in Table 3.8.
Table 3.8 Blocking and antibody incubation
Solution Incubation temperature
Staining reaction and bleaching
Alkaline phosphatase, coupled to antibodies, performs a reaction, in which its substrates BCIP (5-Bromo-4-chloro-3-indolyl phosphate) and NBT (Nitro blue tetrazolium chloride) were oxidized to a blue precipitate, gives a strong staining specifically in the regions, where the RNA probe is present. The staining reaction was performed as follows (Table 3.9).
Table 3.9. Coloring reaction
Solution Incubation time (min)
MAB 5x 5 min
APB 3x 5min
APB with NBT/BCIP Up to three days (until stained)
Upon the staining embryos were fixed in MEMFA, washed with PTw, documented and stored in 100% ethanol. Pigmented embryos undergo bleaching procedure (Table 3.10)
Table 3.10. Bleaching of embryos
Solution Incubation time (min)
2xSSC 3x 5 min
2xSSC with 50% formamide, 1% H2O2, On the light
30min – until bleached, the solution should be replaced each 15 min
MEMFA 30 min
PTw 3x 5 min