Journal:

Article Title: Upregulation of Brain-Derived Neurotrophic Factor by application of Fibroblast Growth Factor-2 to the cut optic nerve is important for long term survival of retinal ganglion cells

doi: 10.1002/jnr.21793

Figure Lengend Snippet: FGF-2 treatment increases the proportion of RGCs that contain BDNF immunostaining. A. Retrograde TDA labeling of RGCs at 6 weeks after axotomy and PBS treatment. B. BDNF immunostaining of the same section as in A, showing approximately 60% of RGCs containing BDNF immunoreactivity (arrows). The inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL) are indicated. Other TDA-labeled RGCs do not contain BDNF (asterisks). C. Retrograde TDA labeling of RGCs at 6 weeks after axotomy and FGF-2 treatment. D. BDNF immunostaining of the same section as in C; almost all RGCs contain BDNF. E. Percentage of RGCs that contain significant BDNF immunoreactivity, with a significant increase after FGF-2 treatment. F. The percentage of RGCs that contain significant TrkB immunoreactivity shows no change with FGF-2 treatment. G. The intensity of BDNF immunoreactivity in stained RGCs significantly increases after FGF-2 treatment. H. The intensity of TrkB immunoreactivity in stained RGCs significantly increases after FGF-2 treatment. Scale bar in A: 30 μm.

Article Snippet: They were then incubated overnight with polyclonal antisera against human BDNF (1:200, Promega, cat. G1541) or mouse full-length TrkB (1:200, Promega, cat. G1561) diluted in 0.1M PBS + 0.3% Triton X-100 + 1% BSA.

Techniques: Immunostaining, Labeling, Staining

Journal:

Article Title: Upregulation of Brain-Derived Neurotrophic Factor by application of Fibroblast Growth Factor-2 to the cut optic nerve is important for long term survival of retinal ganglion cells

doi: 10.1002/jnr.21793

Figure Lengend Snippet: BDNF and TrkB mRNA in RGCs at 6 weeks after axotomy. A. A PBS-treated retina shows some BDNF mRNA in the ganglion cell layer (GCL) and inner nuclear layer (INL). The inner plexiform layer (IPL) is also indicated. B. 6 weeks after axotomy and FGF-2 treatment, more BDNF mRNA is present in the GCL. C. At 6 weeks after axotomy and PBS treatment, low levels of TrkB mRNA are present in the GCL. D. After axotomy and FGF-2 treatment, more TrkB mRNA is present in the GCL. Scale bar in A: 30 μm.

Article Snippet: They were then incubated overnight with polyclonal antisera against human BDNF (1:200, Promega, cat. G1541) or mouse full-length TrkB (1:200, Promega, cat. G1561) diluted in 0.1M PBS + 0.3% Triton X-100 + 1% BSA.

Techniques:

Journal:

Article Title: Upregulation of Brain-Derived Neurotrophic Factor by application of Fibroblast Growth Factor-2 to the cut optic nerve is important for long term survival of retinal ganglion cells

doi: 10.1002/jnr.21793

Figure Lengend Snippet: Cy3-labeled BDNF siRNA applied to the cut axons is transported retrogradely to the RGC cell bodies. A. At 3 days after axotomy, the GCL of a flat-mounted retina shows punctate accumulations of Cy3-labeled siRNA in RGCs. B. The GCL of a control retina with PBS application only shows only background fluorescence. Scale bar in B: 50 μm.

Article Snippet: They were then incubated overnight with polyclonal antisera against human BDNF (1:200, Promega, cat. G1541) or mouse full-length TrkB (1:200, Promega, cat. G1561) diluted in 0.1M PBS + 0.3% Triton X-100 + 1% BSA.

Techniques: Labeling, Control, Fluorescence

Journal:

Article Title: Upregulation of Brain-Derived Neurotrophic Factor by application of Fibroblast Growth Factor-2 to the cut optic nerve is important for long term survival of retinal ganglion cells

doi: 10.1002/jnr.21793

Figure Lengend Snippet: BDNF siRNA inhibits the increase in BDNF mRNA elicited by FGF-2. A. Real-time PCR of retinal mRNA shows that axotomy alone has no effect on BDNF mRNA levels at 48 h after axotomy but induces a significant increase by 1 week. FGF-2 treatment increases BDNF mRNA at both 48 h and 1 week. These FGF-2-induced increases are reduced by siRNA treatment, as is the increase due to axotomy at 1 week. Negative control (NC) siRNA has no effect on mRNA levels at 48 h. B. In situ hybridization shows BDNF mRNA in RGCs at 1 week after axotomy and FGF-2 treatment. The inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL) are indicated. C. SiRNA application reduces BDNF mRNA in RGCs. Scale bar in B: 30 μm.

Article Snippet: They were then incubated overnight with polyclonal antisera against human BDNF (1:200, Promega, cat. G1541) or mouse full-length TrkB (1:200, Promega, cat. G1561) diluted in 0.1M PBS + 0.3% Triton X-100 + 1% BSA.

Techniques: Real-time Polymerase Chain Reaction, Negative Control, In Situ Hybridization

Journal:

Article Title: Upregulation of Brain-Derived Neurotrophic Factor by application of Fibroblast Growth Factor-2 to the cut optic nerve is important for long term survival of retinal ganglion cells

doi: 10.1002/jnr.21793

Figure Lengend Snippet: Application of BDNF siRNA to cut axons reduces the increase in BDNF staining elicited by FGF-2. A. Control retina shows strong BDNF immunostaining in RGCs. The inner nuclear layer (INL), inner plexiform layer (IPL) and ganglion cell layer (GCL) are indicated. B. At 1 week after axotomy, BDNF immunostaining is reduced in RGCs. C. FGF-2 treatment increases BDNF staining in RGCs. D. FGF-2 coupled with siRNA reduces BDNF staining. Scale bar in A: 30 μm.

Article Snippet: They were then incubated overnight with polyclonal antisera against human BDNF (1:200, Promega, cat. G1541) or mouse full-length TrkB (1:200, Promega, cat. G1561) diluted in 0.1M PBS + 0.3% Triton X-100 + 1% BSA.

Techniques: Staining, Control, Immunostaining

Journal:

Article Title: Upregulation of Brain-Derived Neurotrophic Factor by application of Fibroblast Growth Factor-2 to the cut optic nerve is important for long term survival of retinal ganglion cells

doi: 10.1002/jnr.21793

Figure Lengend Snippet: Application of BDNF siRNA to cut axons has no effect on TrkB mRNA. A. Real-time PCR of retinas shows that axotomy and PBS treatment increase TrkB mRNA levels at 48 h and 1 week after axotomy. FGF-2 treatment of the cut axons doubles this increase, at 48 h only. Neither BDNF nor negative control siRNA has any significant effect upon the increases in TrkB mRNA induced by axotomy or by FGF-2. B. In situ hybridization shows TrkB mRNA in RGCs at 48 h after axotomy and FGF-2 treatment. C. SiRNA application has no effect on TrkB mRNA in RGCs. Scale bar in B: 30 μm.

Article Snippet: They were then incubated overnight with polyclonal antisera against human BDNF (1:200, Promega, cat. G1541) or mouse full-length TrkB (1:200, Promega, cat. G1561) diluted in 0.1M PBS + 0.3% Triton X-100 + 1% BSA.

Techniques: Real-time Polymerase Chain Reaction, Negative Control, In Situ Hybridization

Journal:

Article Title: Upregulation of Brain-Derived Neurotrophic Factor by application of Fibroblast Growth Factor-2 to the cut optic nerve is important for long term survival of retinal ganglion cells

doi: 10.1002/jnr.21793

Figure Lengend Snippet: Application of BDNF siRNA to cut axons reduces the long term survival effect on RGCs elicited by FGF-2. A. Quantification of numbers of retrogradely-labeled RGCs at 6 weeks after axotomy. Axotomy alone leads to a 40% decrease in RGCs. Cell numbers decrease further with BDNF siRNA application to the cut axons. Negative control siRNA is not different from PBS alone. FGF-2 application to the cut axons rescues RGCs. Application of BDNF siRNA along with FGF-2 abolishes this survival effect. B-F. Representative flat mounts of retinas showing RGCs retrogradely labeled with axonally applied TDA with the different treatments. Scale bar in E: 50 μm.

Article Snippet: They were then incubated overnight with polyclonal antisera against human BDNF (1:200, Promega, cat. G1541) or mouse full-length TrkB (1:200, Promega, cat. G1561) diluted in 0.1M PBS + 0.3% Triton X-100 + 1% BSA.

Techniques: Labeling, Negative Control