3 Methods and materials
3.6 Histological methods
3.6.1 Solutions (in alphabetical order)
- AP buffer: 100mM Tris/HCl (pH 9.5), 50mM MgCl2, 100mM NaCl, 0.1% Tween 20, 5mM Levamisole.
- AP staining solution: 4.5l NBT, 3.5l BCIP in 1ml AP buffer.
- A-PBS: 103mM NaCl, 2.7mM KCL, 0.15mM KH2PO4, 0.7mM NaH2PO4 pH7.5
Figure 9: Western blot detection of CHD4-Myc protein
Western blot detection of CHD4-Myc protein to evaluate the efficiency of CHD4 Morpholinos to reduce CHD4 protein translation in vitro. Increasing amounts of CHD4 Morpholinos reduced the in vitro translation of CHD4 protein.
- A-PBS-T: APBS with 0.1% Tween20
- Blocking buffer: PBT plus 10% heat inactivated serum
- Citrate buffer: Stock A: 0.1M citrate monohydrate (10.5g for 500ml solution) - Stock B: 0.1M Trisodiumcitrate-dihydrate (14.7g for 500ml solution)
- Working Sol.: 9ml A with 41ml B to 450 ml ddH2O. pH should be 6. - DAPI: Hoechst dye (1mg/ml) 1:1000 in APBS-T
- Dent’s Fixative: 80% methanol, 20% dimethyl sulfoxide (DMSO)
- Elvanol: 2.4g Moviol 4 (Hoechst, Frankfurt) were mixed with 6g glycerol and 6ml ddH2O and stirred at least for 2h at room temperature. Then 2ml 0.2M Tris/HCl pH8.5 were added and incubated for 10min at 60°C. Afterwards 50mg/ml DABCO (1.4-Diazabicyclo(2.2.2) Octane) were added and centrifuged for 30 min at 17000 x g. Aliquots of the supernatant were stored at -20°C.
- MEMFA: 0.1M MOPS, 2mM EGTA, 1mM MgSO4, 3.7% formaldehyde (pH7.4 at 23°C), prepare freshly.
- PBS: 137mM NaCl, 2.7mM KCl, 8mM Na2HPO4, 1.7mM KH2PO4 (pH7.2 at 23°C).
- PBT: PBS, 2mg/ml BSA, 0.1% Triton-X-100.
3.6.2 Immunocytochemistry
For immunocytochemistry, the first step was to remove the vitelline membrane from the embryos. Subsequently, the embryos were fixed in MEMFA for 1-2h at room temperature with rotation. After rinsing with PBS, the embryos were gradually dehydrated with methanol and incubated in methanol at -20°C over night. Rehydration was performed by 80%, 50%, 0% methanol in PBS for 5 min each, followed by a 5min rinse with PBS and one rinse in PBT for 15min. Unspecific antibody binding sites were blocked by incubating the embryos in PBT plus 10% heat inactivated goat serum at room temperature for 1h. The primary antibody was diluted in the blocking buffer and incubated over night at 4°C. Afterwards the embryos were washed 6 times with PBT for one hour. The secondary antibody solution, consisting of the secondary antibody, coupled with alkaline phosphatase, diluted 1:1000 with blocking buffer was added to the embryos and was incubated over night at 4°C. Subsequently, the embryos were washed 6 times with PBT for on hour. Prior to staining, the endogenous alkaline phosphatases were blocked by the addition of Levamisol to the AP buffer. The embryos were incubated twice in AP buffer for
30min. Staining was performed in 1 ml staining solution in the dark for 30 to 120min at room temperature. The staining reaction was stopped by washing the embryos in PBS to titrate out the staining solution. The stain was fixed in MEMFA over night. Bleaching of the embryos was reached by washing in bleaching solution for 2h on a light box.
3.6.3 Antibodies
3.6.3.1 Primary antibodies
Against:
xCHD4 as GST-fusion protein of the N-terminus (aa1-aa357) (Linder et al, 2007; Singhal, 2005):
CH4-N 3A11: subtype IgG2a (WB negative, positive in IP + ChIP, ICC negative) CH4-N 5H4: subtype IgG1 (WB negative, positive in IP + ChIP, ICC negative) CH4-N 5A2: subtype IgG2a (WB negative, positive in IP + ChIP, ICC negative) Antigen xCHD4 as GST-fusion protein of the C-terminus (aa 1513- aa 1891) (Linder et al, 2007)
CH4-C 7C9: subtype IgM (WB negative, positive in ChIP, ICC positive) CH4-C 7E8: subtype IgM (WB negative, positive in ChIP, ICC positive).
For the production of rat monoclonal antibodies, the C-terminal domain of xCHD4 (amino acids 1513-1891) was cloned into the pGEX-4T3 bacterial expression vector (Amersham), expressed in Escherichia coli, and purified. The antibodies were generated in collaboration with Dr. Elisabeth Kremmer, GSF Munich. The GST-fusion proteins were cloned and purified in our laboratory by Dr. rer. nat. Kathrin Mansperger. Positive primary hybridoma cell supernatants were prescreened by the Kremmer laboratory concerning their specificity to bind the antigen, but not to the GST-fusion part. Using Western blot and immunoprecipitation (IP) analyses, positive clones were further analyzed for their specific detection of in vitro translated antigens in our lab. Subsequently, the Kremmer laboratory stabilized the positive tested hybridoma clones. Clones were then tested in IPs and ChIP-IPs for their specificity and affinity to their antigen.
Dr. Elisabeth Kremmer
Helmholtz Zentrum München, Serviceeinheit Monoklonale Antikörper Institut für Molekulare Immunologie
Tel.: 089/7099-321 (office) or -318 (laboratory), Fax: 089/7099-300 e-mail: [email protected]
Caspase3: (Upstate Biotechnology, Lake Placid, NY) H3S10Ph: (Upstate Biotechnology, Lake Placid, NY) H3S28P: (Upstate Biotechnology, Lake Placid, NY)
3.6.3.2 Secondary antibodies
Cy3 Indocarbocyanin, donkey anti-rat, Dianova, 1:200 diluted
Fluorescein- Isothiocyanat (FITC), donkey anti-mouse, Dianova, 1:200 diluted AlkPhos, Fab Ig, sheep anti mouse, Chemicon, 1:1000 diluted
3.6.4 Immunofluorescence
For immunofluorescence analysis of whole embryos, the same protocol was used with exception that no blocking of endogenous phosphatase, fixation of staining and bleaching was performed or necessary.
3.7 Confocal microscopy
Series of confocal sections through whole-mount embryos, focused on the central nervous system were collected with a Leica SP5 microscope equipped with a Plan/Apo 63X 1.4 NA oil immersion objective. For each optical section, images (512*512 pixels) were collected sequentially for three fluorochromes. The stacks of each sectios at 4 micrometers were recorded as separated eight-bit grayscale Z- planes with voxel size 120x120x200 nm (XxYxZ). The “Abi prism” software was used to convert .lsm (laser-scanning-microscopy) files, obtained by the confocal microscopy, to .tif files to be processes by ImageJ software. RGB stacks were reconstructed with the three channels function of ImageJ. The mean integrated fluorescence intensity was quantified with ImageJ according to the following steps:
1) Open File of two-dimensionally reconstructed scan:
2) Image – Type – RGB Stack – Make Montage:
3) Image - Adjust - Thresholds:
4) Analyze - Set Measurements, 5) Creating a Mask