Pth4:eGFP transgenic zebrafish embryos at 1 and 2 dpf were fixed with 4% PFA and washed in PBST. They were then washed in PBDT (1% BSA, 1% DMSO, 0.1% Triton X-100 in PBS, pH 7.4), blocked in 10% normal goat serum/PBDT, and incubated overnight at 4°C with primary antibodies to HuC/D (1/100) and GFP (1/400, Abcam ab6673). Further PBST washes and blocking were followed by secondary antibodies overnight at 4°C. Hoechst 34580 was added to stain nuclei (1/2500). After further PBDT and PBS washes, embryos were mounted for confocal imaging.

Abbreviation: e, eye; hy, hypothalamus; m, midbrain; sc, spinal cord. Scale bars: 100 μm (A-C) 50 μm (D-G).

In utero electroporation of Disc1 and Disc1-100P constructs into wild-type neocortex and analysis at P21.

(Panels D-E”) Expression of the constructs was assessed.

(Panels D-D'') 2 days after transfection in vitro.

(Panels E-E'') at P21 in vivo.

Immunochemistry for FLAG and GFP showed that constructs encoding either WT Disc1, the Disc1-100P variant, or GFP alone, expressed these protein species in transfected HEK-293 cells in vitro (Fig 5D–5D”) and in P21 postmitotic cortical neurons in vivo (Fig 5E–5E”)

Immunofluorescence for assessment of GFP⁺ myofibers in rat tissue.

VML affected muscle from the 50% MG + HA+LMN group were probed for the presence of GFP. GFP+ fibers were detected in a qualitatively similar magnitude at both 2 and 8 weeks post-injury indicating viable engraftment of donor derived muscle progenitor cells. Scale bars are 1mm for whole mount images, 50 μm for regions of interest.

A portion of the TA muscle from the defect region was embedded in a talcum-based gel, frozen in 2-methylbutane, and supercooled in liquid nitrogen. Cryosections (8 μm) were prepared and stained using standard protocols for hematoxylin & eosin.

ab6673 used at a 1/100 dilution.

Mouse small intestines were washed with DPBS and fixed overnight at 4°C in Zinc formalin. Following sectioning and tissue deparaffanization, antigen retrieval was performed with 10mM Tris base (pH 9.0) buffer using a pressure cooker.

For immunohistochemistry, sections were quenched of endogenous peroxidases by 3% H₂O₂, and sequentially blocked with Avidin D, biotin, and protein blocking reagents. Primary antibody incubation was conducted at 4°C overnight. Secondary biotinylated antibody was added at a dilution of 1/200, and incubated 2 hours at room temperature. Finally, sections were stained according to the ABC peroxidase protocol and counterstained with hematoxylin.

ab6673 used at a 1/200 dilution.

Panel D: Representative anti-eGFP immunofluorescence of macroH2A WT and DKO jejunum counterstained with DAPI (blue).

All lanes : Anti-GFP antibody (ab6673) at 1 µg/ml (o/n at 4degC)

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) lysate at 10 µg
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) lysate at 10 µg
Lane 3 : CHO/K1 lysate at 10 µg
Lane 4 : MDA-MB-231 (Human breast adenocarcinoma cell line) lysate at 10 µg
Lane 5 : A431 (Human epidermoid carcinoma cell line) lysate at 10 µg
Lane 6 : Jurkat (Human T cell leukemia cell line from peripheral blood) lysate at 10 µg
Lane 7 : NIH/3T3 (Mouse embryo fibroblast cell line) lysate
Lane 8 : E-coli HCP control, 50 ng
Lane 9 : FLAG Positive control lysate at 10 µg
Lane 10 : Red fluorescent protein, 50 ng
Lane 11 : Green fluorescent protein, 50 ng
Lane 12 : Glutathinoe-S-Transferase protein, 50 ng
Lane 13 : Maltose Binding protein, 50 ng

Secondary
All lanes : Peroxidase goat secondary antibody, 60 min at RT at 1/30000 dilution

Blocking Buffer: 1% Casein-TTBS for 30 min at RT.

E5.5 Hex-GFP transgenic mouse embryo stained for GFP using ab6673 at 1/500 dilution. Secondary antibody is a fluorochrome conjugated anti-goat IgG secondary antibody at 1/10,000 for 45 min at RT.

Staining: GFP as green fluorescent signal with DAPI blue counterstain.

All lanes : Anti-GFP antibody (ab6673) at 1 µg/ml

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cells
Lane 2 : Mock transfected HeLa cell lysate

Lysates/proteins at 35 µg per lane.

Secondary
All lanes : IRDye® 800 conjugated Donkey-a-Goat IgG [H&L] at 1/2500 dilution

Additional bands at: 33 kDa. We are unsure as to the identity of these extra bands.

All lanes : Anti-GFP antibody (ab6673) at 1/1000 dilution

Lane 1 : MRC5VA lung fibroblast whole cell lysate overexpressing EGFP alone
Lanes 2-3 : MRC5VA lung fibroblast whole cell lysate overexpressing an EGFP fusion protein

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : HRP-conjugated anti-goat polyclonal at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Observed band size: 27,55 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

See Abreview

Immunofluorescence of TGN mouse liver labeling GFP on hepatocytes with ab6673.

Immunohistochemistry of GFP transgenic mouse liver labeling GFP with ab6673.