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. 2023 Jan:226:109310.
doi: 10.1016/j.exer.2022.109310. Epub 2022 Nov 15.

Repeat Brn3a immunolabeling rescues faded staining and improves detection of retinal ganglion cells

Ryan E Shindler  1 , Jipeng Yue  1 , Brahim Chaqour  1 , Kenneth S Shindler  2 , Ahmara G Ross  3
Affiliations

Repeat Brn3a immunolabeling rescues faded staining and improves detection of retinal ganglion cells

Ryan E Shindler et al. Exp Eye Res. 2023 Jan.

Abstract

Immunofluorescence is used in numerous research areas including eye research to detect specific antigens in cells and tissues. One limitation is that fluorescent signal can fade, causing detection problems if data recording was not completed in a timely manner or if additional data acquisition is required. The ability to repeat immunostaining for the same antigen after initial fluorescence has faded may require time-consuming and potentially damaging steps to remove primary antibodies. Our studies assessed whether immunofluorescence could be reapplied to previously labeled retinal ganglion cells (RGCs). To examine whether immunostaining of Brn3a, a commonly used RGC marker, could be repeated in retinas with previously faded immunostaining, retinal whole mounts were labeled with anti-Brn3a primary antibodies and green fluorescent secondary antibodies, then allowed to fade over time. Faded retinas were restained with anti-Brn3a antibody followed by secondary antibody, or with secondary antibody alone. Results show restaining with anti-Brn3a primary antibody followed by Alexa-fluor green secondary antibody is effective for RGC detection. Repeat RGC labeling improved the clarity of staining compared with original staining prior to fading, with significant reduction in the percentage of blurry/out of focus fluorescent cells (6 vs 26%); whereas, repeat application of secondary antibody alone was not effective. Preflattening retinas under a coverslip prior to initial Brn3a staining also increased the clarity of staining, and facilitated significantly more accurate automated counting of RGCs. Findings suggest Brn3a antigen remains accessible for repeat immunofluorescence labeling after original staining fades. Staining retinas after flattening tissue may enhance the clarity of staining and accuracy of automated RGC counting. Repeat immunofluorescence staining, without the need to strip off prior bound antibodies, may be useful in other tissues as well and warrants future examination.

Keywords: Brn3a staining; Immunofluorescence; Retinal ganglion cells.

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Conflict of interest statement

Declaration of competing interest The authors declare no conflicts of interest related to this work.

Figures

Figure 1.
Figure 1.
Representative photographs of retinas after original Brn3a staining, faded Brn3a staining, repeat Brn3a staining, and attempted secondary antibody repeat staining. Original Brn3a staining (A) shows numerous antigen positive cells. Faded staining (B) has no clearly defined cells. The restained retina (C) shows many Brn3a-postive cells, similar to the original stained slides. The secondary antibody only re-stained retina (D) is similar to the faded staining picture with no detectable signal. There is no significant difference in the average number of Brn3a-positive cells counted in standardized retinal fields between original stained and restained retinas (N = 6 retinas/group; p = 0.5858). Scale bars = 25 microns.
Figure 2.
Figure 2.
Representative photo of original Brn3a (A) staining, which has many cells both clear and out of focus; repeated Brn3a staining (B), which has many cells, most of which are clearly defined; and a retina pre-flattened under a coverslip for one week prior to original Brn3a staining (C), which also shows numerous cells in clear focus. (D) Comparison of percentages of blurry cells from original staining (N = 6 retinas), repeat staining (N = 6), and original staining after preflattening (N = 6) shows that significantly fewer RGCs are out of focus in re-stained (**p < 0.01) and pre-flattened (***p < 0.001) retinas as compared with original Brn3a staining. Scale bars = 25 microns.
Figure 3.
Figure 3.
Preflattening retinas before staining improves concordance between human and automated cell counts. (A) Representative images of one original, one restained and one preflattened retina after staining for Brn3a showing respective image orientation manipulations: rotation 180 degrees clockwise and mirroring across the vertical meridian. Images were taken at 40x magnification and mixed in random order with images from other retinas before being counted by masked individuals. One of four images from each staining group is shown. (B) The standard error of the number of Brn3a-positive cells counted by each counter (human or automated) was calculated for each set of 3 duplicate/inverted images as shown in A. Seven masked human counters varied in their individual counts of three duplicate images (N = 4 sets of 3 images/group) by no more than ±3 cells regardless of whether retinas were originally stained, restained, or preflattened before staining. Simple RGC automated counting was also highly consistent with no more than ±1 cells between counts of 3 copies of each image regardless of whether retinas were originally stained, restained, or preflattened before staining (N = 4 sets of 3 images/group). (C) Differences in automated vs. manual counters are displayed as the percent difference between the automated Simple RGC count and each individual human counter (N = 7). Percent difference was calculated by subtracting automated means from the mean of each human counter and dividing by the human mean. The percent difference between automated and manual counts in original stained retinal images was significantly higher (***p<0.001) than the percent differences between automated and manual counts in restained or preflattened retinal images.
Figure 4.
Figure 4.
Illustration shows potential explanation for observed improvement in clarity of Brn3a staining after retinas have flattened over time under a coverslip prior to staining or restaining. Dissected retinas are flat-mounted on slides but tissue retains some thickness and microscopic folds/undulations (top) that can flatten further over time under a coverslip (bottom).
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