Summary of the three approaches.

<p>Summary of the three approaches.</p

Intact ON labelling approach is also feasible for GB filling of RGCs, with increased stability compared to ON cut approach.

<p>4A, B, and C represent ON cut approach with GB labelling at 7 days, 2 weeks and 3 weeks; 4 D, E, and F represent intact ON approach with GB labelling at 7 days, 2 weeks and 3 weeks, respectively. Scale bar represents 50 μm.</p

FG application onto the surface of ON could be taken up by intact ON fibers.

<p>5A: Cross section of FG labelled ON; 5B: Longitudinal section of FG labelled ON; 5C: Cross section of ON through sclera approach; 5D: Picture of sclera approach labelled retina. Scale bar represents 50 μm.</p

Decreased FG labeling without RGC loss at prolonged time points.

<p>A: FG labeling with intact ON approach at 7 days. B: IBA1 immunohistochemistry showing microglial cells. C: beta-tubulin immunohistochemistry showing all RGCs and axons. D: merged image of A and C. Arrows in C and D are showing RGCs without FG filling. Scale bar represents 50 μm.</p

Intact ON labelling approach leads to stable filling without subsequent RGC loss.

<p>3A, B, C, and D represent FG labelling of RGCs through ON cut approach at 2 days, 7 days, 2 weeks and 3 weeks. 3E, F, G, and H represent FG labelling of RGCs through intact ON approach at 2 days, 7 days, 2 weeks and 3 weeks, respectively. Arrows in C and D are showing residual RGCs that are with big soma size (potentially alpha-RGCs). Scale bar represents 50 μm.</p

RGCs numbers per mm² using different labelling methods.

<p>Note:</p><p>^# for non-significant.</p><p>* For P<0.05 in compared to SC labeling.</p><p>RGCs numbers per mm² using different labelling methods.</p

Intact ON labeling results in minimal injury to ON.

<p>7A, B: normal ON. 7C: 4 days after intact labeling. 7D: 7 days after intact labeling. Arrows in C and D indicate the myelin damage. Scale bar represents 10 μm.</p

Intact ON labelling approach results in the same quality of RGC filling with fluorescent dyes.

<p>2A: FG labelling of RGCs through superior colliculus application; 2B: FG labelling of RGCs through ON cut approach; 2C: FG labelling of RGCs through intact ON approach; 2D: GB labelling of RGCs through intact ON approach. Scale bar represents 20 μm.</p

Fluoro-Gold (FG) application onto the surface of the optic nerve (ON) is sufficient to label all RGCs at 2 days.

<p>1A: FG labelling of RGCs through superior colliculus application after 2 days; 1B: FG labelling of RGCs through ON cut approach after 2 days; 1C: FG labelling of RGCs through intact ON approach after 2 days; 1D: Granular Blue (GB) labelling of RGCs through intact ON approach after 2 days. Scale bar represents 100 μm.</p

Figure 2

<p>Drawing of the olfactory bulb (A) and the location of the injury and the sampling area. (B): Photomicrograph of the sagittal section of olfactory bulb. The sample grid used for every animal includes both the yellow and red areas and were further broken down into Locations I, II, and III. This was to show the movement and depletion of the microglia from these areas and their subsequent return. Sample grid: 150 µm.</p