A New Method to Visualize the Intact Subretina From Retinal Pigment Epithelium to Retinal Tissue in Whole Mount of Pigmented Mouse Eyes

Abstract

PurposeThe subretinal layer between apical retinal pigment epithelium (RPE) and the apices of the photoreceptor outer segments is important to aging and degenerative pathogenesis, but current protocols do not provide intact horizontal images of this retinal space. Thus, an RPE/retina whole mount staining protocol was developed to observe integral subretinal regions.MethodsRPE/retina whole mounts were stained instead of separated retina or RPE whole mounts. Hydrogen peroxide (H2O2) treatment was applied in different conditions of concentration, time, and temperature for the bleaching of RPE and choroidal melanocyte pigmentation in the pigmented RPE/retina whole mounts before antibody staining.ResultsAn RPE/retina whole mount staining protocol provided better morphology of the photoreceptor outer segment than current retina whole mount. For the pigmented eyes, 10% H2O2 pretreatment effectively bleached melanin at 55°C less than 2 hours, or at 4°C within 7 days, without significant effect on immunolabeling efficacy of most antibodies tested. Actually, 55°C bleaching improved immunolabeling intensities compared to the nonbleaching control. This melanin bleaching RPE/retina protocol was applied further to observe macrophage/microglia extending from the sclera to outer plexiform layer in the CX3CR1+/GFP retina.ConclusionsThe pigment bleaching RPE/retina whole mount allowed integral horizontal imaging between choroid to photoreceptor layers, which could not be accomplished with existing methods of separated retina or RPE whole mount. Under these procedures, antigenicity of most antibodies also was well preserved.Translational RelevanceThis efficient protocol provides a tool to observe an integral view of subretinal structures including macrophage/microglia and transplanted cells, and further allows study of the interrelationship between the choroid and photoreceptor in models of aging, disease, and retinal degeneration.

Visualization of the Retina in Intact Eyes of Mice and Ferrets Using a Tissue Clearing Method

“…Previously, it was reported that the treatment of the retinal pigmented epithelium and choroidal melanocytes with H 2 O 2 eliminated black color efficiently. 21 We therefore hypothesized that treatment of eyes with H 2 O 2 and CUBIC would make the eyes of C57BL/6J mice transparent. We tried several concentrations, incubation times, and temperatures for the H 2 O 2 treatment and found that 10% H 2 O 2 at 55°C for 3 hours was optimum for eliminating black color from intact eyes of C57BL/6J mice ( Figs.…”

“…The pigmented eyes were immersed in 10% H 2 O 2 /PBS for 3 hours at 55°C to bleach the melanin. 21 The eyes were washed with 0.5% Triton X-100/PBS three times for 10 minutes each at room temperature.…”

Peroxisome turnover and diurnal modulation of antioxidant activity in retinal pigment epithelia utilizes microtubule-associated protein 1 light chain 3B

“…Sections were washed 3x in 1x PBS and bound primary antibodies were detected by incubation with Alexa-fluor conjugated secondary antibodies diluted in blocking buffer (Thermo Fisher/Invitrogen, Eugene, OR) for 1 hr at 37°C. For flat mounts of RPE/choroid/sclera, melanin and other pigment was bleached after fixation, using the method of Kim and Assawachananont (38). Briefly, each eyecup was incubated at 55° C in 10% peroxide/1x PBS for 2.5 hr and immunolabeled as above after 3-4 washes in PBS.…”

Expression, regulation and function of miR-126 in the mouse choroid vasculature

“…For flat mount staining of the choroidal vasculature, pigment bleaching in RPE cells and choroidal melanocytes was performed as described (Kim and Assawachananont, 2016). Briefly, pigmented eyes were fixed in 4% paraformaldehyde PBS for 30 min to 2 h. After washing with PBS three times, the cornea and lens were removed before 3% hydrogen peroxide bleaching at 55 °C for 30 min to 2 h depending on the stage, or at 4 °C for several days.…”