Z1 apotome microscope

Fluorescent signals were studied with a Zeiss LSM780 confocal microscope. High-resolution images were captured using a 20×/0.8 NA objective, a 0.6–1× optical zoom, and the Zen software (Carl Zeiss). Different fluorochromes were detected with laser lines 488 nm for FITC and AF488 and 561 nm for Cy3. Emission filters were 493–556 nm for FITC and AF488 and 570–624 nm for Cy3. To prevent emission crosstalk between the fluorophores, the red channel was recorded separately from the green one (‘smart setup’ function). To illustrate the results, confocal Z-stacks (Z-steps: 0.85–1 μm; pixel dwell time: 0.79–1.58 μs; resolution: 1024 × 1024 pixels; pinhole size: set at 1 Airy unit) were merged using maximum intensity Z-projection (ImageJ). The final figures were adjusted in Adobe Photoshop using the magenta-green color combination and saved as TIF files.
Fetal sections were examined using an Axio Imager.Z1 ApoTome microscope (Carl Zeiss, Germany) equipped with a motorized stage and an AxioCam MRm camera (Zeiss). For confocal observation and analyses, an inverted laser scanning Axio observer microscope (LSM 710, Zeiss) with an EC Plan NeoFluorÅ ~100/1.4 numerical aperture oil-immersion objective (Zeiss) was used (Imaging Core Facility of IFR114, of the University of Lille, France).

Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.5% TritonX-100/PBS for 20 min. After washing with PBS, slides were incubated with 100 ug/ml RNase A for 1 h and 40 units/ml pepsin for 10 min at 37 °C, and dehydrated for 2 min each in 70%, 80% and 100% ethanol. Slides were denatured in 70% formamide for 5 min at 73 °C. DNA probes were prepared and labeled with rhodamine-dUTP and fluorescein-dUTP. Probes were denatured for 5 min at 73 °C and hybridized to the denatured slides for 24 h at 37 °C. Slides were washed for 5 min each in 50% formamide/2x SSC and 0.1% Tween 20/2x SSC at 42 °C, dehydrated for 2 min each in 70%, 80% and 100% ethanol, and mounted on glass slides. FISH signals were detected by a Zeiss imager Z1 Apotome Microscope. DNA probes used in these assays were the BAC RP11–599H8 (155 kb), RP11–942D19 (182 kb), BAC RP11–956H14 (185 kb) and RP11–96K4 (168 kb). Interprobe distances under each condition were calculated as described previously50 (link). 100 cells (200 loci) were used to measure interprobe distances for each condition, and the statistical significance of differences between the indicated groups was measured by the Mann-Whitney U test.

Fluorescence microscopy was performed with a Z1 Apotome microscope equipped with a Zeiss Axiocam digital camera (Zeiss, Oberkochen, Germany). Images were captured using Zen software (Zeiss) and the Z-stack function (14-bit depth, 2752*2208 pixels, pixel size = 0.227 µm, 9 Z-planes at 1 µm steps). The raw images were converted into maximum intensity projections (MIF) using Zen software and saved as TIFF files.
Photoreceptors stained by the TUNEL assay were counted manually on three images per explant, the average cell number in a given ONL area was estimated based on DAPI staining and used to calculate the percentage of TUNEL-positive cells. Cones stained with Arr3 were counted on Apotome images using MIF. Cone numbers are expressed as Arr3-positive cells visible in 100 µm stretches of retinal circumference.
Adobe Photoshop CS6 (Adobe Systems Inc, San Jose, CA, USA) and Adobe Illustrator CC 2019 software was used for primary image processing. All data given represent the means and standard deviation from at least five different animals. Statistical comparisons between experimental groups were made using Student’s paired t-test or ANOVA and multiple comparisons correction with Tukey’s post hoc test (Figure 1) using GraphPad Prism 9.1 for Windows (GraphPad Software, La Jolla, CA, USA). Levels of significance were: *=p<0.05; **=p<0.01; ***=p<0.001.

After the end of last recording sessions, rats were deeply anesthetized with pentobarbital sodium (150 mg/kg, i. p.) and transcardially perfused with 0.01 M phosphate buffer, pH = 7.2, in a 0.9% (w/v) saline solution, followed by 4% paraformaldehyde. Brains were removed, post fixed in 4% paraformaldehyde for 4 h and stored in 30% (w/v) sucrose solution before they were frozen, and later sectioned into 30 µm slices. Brain slices were used to locate optrode or optical fiber end position, and to quantify opsins expression in the tissue. The final location coordinates were identified using a rat brain atlas [29 ]. Stained sections were observed using a Zeiss Z1 Apotome microscope. Only rats with correct location of the implanted optrode and optical fiber, as well as sufficient viral expression were included in the data analysis.

For immunohistochemistry analysis, tissue preparation, mounting, and blocking were carried out as previously described [27 (link)]. Slides were stained with primary antibodies specific for C1q (Abcam, Cambridge, UK, ab1822451) or PSD-95 (Abcam Cambridge, UK ab13552) overnight, washed three times in Tris-buffered saline (TBS), and stained for the secondary antibody, goat anti-rabbit Alexa-568 (Invitrogen, Carlsbad, CA, USA, A-11011) or goat anti-mouse Alexa-488 (Goat anti-mouse 488 Invitrogen, Carlsbad, CA, USA, A11001). Tissues were fixed using ProLong Gold (Invitrogen, Carlsbad, CA, USA, P36930) and a standard slide cover sealed with nail polish. Two to five images separated by 50–100 µm in the dorsal hippocampus were averaged per animal. For C1q staining, z-stack images were acquired on a Zeiss Imager.Z1 Apotome microscope (Zeiss, Thornwood, NY, USA) controlled by ZEN software (Zeiss 2012, Thornwood, NY, USA) with 200× magnification (C1q). For PSD-95 staining, z-stack images were acquired on a Nikon High-Speed Widefield Confocal microscope (Ti inverted fluorescence; CSU-W1, CSU-W1, Melville, NY, USA) at the UCSF Nikon Imaging Center with 630× magnification.