Kothurkar AA et al. (2024), Iterative Bleaching Extends Multiplexity (IBEX)...

XB-ART-61067

J Cell Sci 2024 Nov 14; doi: 10.1242/jcs.263407.

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Iterative Bleaching Extends Multiplexity (IBEX) facilitates simultaneous identification of all major retinal cell types.

Kothurkar AA , Patient GS , Noel NCL , Krzywańska AM , Carr BJ , Chu CJ , MacDonald RB .

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To understand the multicellular composition of tissues, and how it is altered during development, ageing and/or disease, we must visualise the complete cellular landscape. Currently, this is hindered by our limited ability to combine multiple cellular markers. To overcome this, we adapted a highly multiplexed immunofluorescence (IF) technique called Iterative Bleaching Extends Multiplexity (IBEX) to the zebrafish retina. We optimised fluorescent antibody micro-conjugation to perform sequential rounds of labelling on a single tissue to simultaneously visualise all major retinal cell types with 11 cell-specific antibodies. We further adapted IBEX to be compatible with fluorescent transgenic reporter lines, in situ hybridisation chain reaction (HCR), and wholemount immunofluorescence (WMIF). We applied IBEX at multiple stages to study the spatial and temporal relationships between glia and neurons during retinal development. Finally, we demonstrate the utility of IBEX across species by testing it on the turquoise killifish (Nothobranchius furzeri) and African clawed frog (Xenopus laevis) to glean large amounts of information from precious tissues. These techniques will revolutionise our ability to visualise multiple cell types in any organism where antibodies are readily available.

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	Fig. 1. The retina is made up of highly organised layers composed of neurons and glia. (A) Schematic of the retina showing the layers and major cell populations with each cell type colour coded. (B) DAPI staining showing the nuclear layers of the retina. (B′) Magnification of B. (C–G) Antibody staining for the main cell types in the zebrafish retina. Arrows in G show horizontal cells. OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. All images representative of three experimental repeats. Scale bars: 25 µm (whole retina images); 10 µm (magnified images).
	Fig. 2. Schematic of the IBEX method. Slides are coated with chrome alum gelatin to prevent tissue loss, then tissue is sectioned onto slides. Antibodies are micro-conjugated by mixing the primary antibody with a linker and fluorophore. The antibodies are applied to the slide, incubated, imaged, then bleached using bright light and lithium borohydride before being re-stained. After the imaging rounds are completed, nuclear stains (DAPI) are used to register the image, allowing for all stains to be visualised together. Created in BioRender by Noel, N., 2024. https://BioRender.com/e79p158. This figure was sublicensed under CC-BY 4.0 terms.
	Fig. 3. Direct conjugation to fluorophores facilitates multiple single species antibody labels on the same tissue. (A) Merged epifluorescence images of a single sagittal retinal section immunolabelled with four antibodies raised in rabbit: PKC-β (magenta), GNAT2 (green), RLBP1 (yellow), Ribeye-A (cyan) and nuclear stain DAPI (grey). (B) Merged image without DAPI. (C) Retinal section immunolabelled with PKC-β, marking bipolar cells. (D) Retinal section immunolabelled with GNAT2 marking cones. (E) Retinal section immunolabelled with RLBP1, marking Müller glia cells. (F) Retinal section immunolabelled with Ribeye-A marking ribbon synapses. All images representative of three experimental repeats. Scale bars: 25 µm.
	Fig. 4. IBEX enables simultaneous labelling of all major retinal cell types. (A) Confocal images of 5 dpf Tg(tp1:eGFP:CAAX) zebrafish retina showing different rounds of immunolabelling (R1–R4) using IBEX and the merge composite image of each of these rounds. R1 was carried out using Alexa Fluor secondary antibodies, whereas other rounds used microconjugated antibodies. (B) Confocal images showing each antibody used in A to immunolabel a single sagittal retinal section with DAPI and 11 different markers: Ribeye-A (dark blue), carbonic anhydrase (CA1, purple), glutamine synthetase (GS, red), PKC-β (green), Zrf-1 (cyan), HuC/D (orange), GFP transgene (yellow), calbindin (CalB, magenta), peripherin-2 (PRPH2, light blue), GNAT2 (orange) and RLBP1 (pink). All images representative of three experimental repeats. Scale bars: 25 µm.
	Fig. 5. IBEX is compatible with fluorescent in situ hybridisation chain reaction. (A) Confocal images of retinal sections showing mRNA expression of cyp26a1, glula, and vsx1, using in situ HCR. (A′–A⁗) Magnification of the region of interest indicated in A. (B–B‴) Confocal images showing reduced signal of Alexa Fluor 488 and Alexa Fluor 647, but not Alexa Fluor 555 after LiBH4 treatment. (B⁗) Heating in sodium citrate at 60°C causes inactivation of Alexa Fluor 555 as well as Alexa Fluor 488 and Alexa Fluor 647. (C) Confocal images of retinal sections immunolabelled with CA1 (yellow), PKC-β (green) and GS (magenta). (C′–C‴) Magnification of the region of interest indicated in C. (D) SimpleITK registered image, showing overlay of both rounds of imaging, and overlay of in situ probes and antibodies detecting Müller glia and bipolar cells, respectively. (D″–D‴) Magnification of the region of interest shown in D and D′. GCL, ganglion cell layer; IPL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer. All images representative of three experimental repeats. Scale bars: 25 µm (whole retina); 10 µm (magnified images).
	Fig. 6. Whole-mount IBEX facilitates whole tissue labelling in zebrafish. (A,A′) Confocal images of whole-mount zebrafish larvae at 5 dpf immunolabelled with GS (orange) and blue opsin (magenta). (B) Tissue after bleaching with LiBH4 showing reduced signal of the fluorophores CoraLite 488 and CoraLite 647. (C,C′) Confocal images of the second round of immunolabelling to detect Zrf-1 (cyan) and GNAT2 (green). (D) Merge of both rounds of immunolabelling using SimpleTK registration pipeline counterstained with DAPI (grey). (E) Overlap of MG markers GS and Zrf-1 across round 1 and 2. (E′) Overlap of photoreceptor markers blue opsin and GNAT2 across round 1 and 2. (F) Magnification of area highlighted in D, showing overlay of MG and photoreceptor labelling. MG, Müller glia. All images representative of three experimental repeats. Scale bars: 25 µm (whole retina); 10 µm (magnified images).
	Fig. 7. Visualisation of glial and neuronal development in the zebrafish retina. Confocal images of the developing zebrafish retina from 2 dpf to 5 dpf immunolabelled with DAPI and seven different markers using IBEX over 3 rounds of immunolabelling with IBEX. A,B,C,D show a merge of all seven markers with nuclear stain DAPI; A′,B′,C′,D′ show the same overlay of markers without DAPI. A″,B″,C″,D″ show the first two rounds of immunolabelling of bipolar cells (PKC-β, green), glial intermediate filaments (Zrf-1, cyan), horizontal cells (CA1, purple) and Müller glia (GS, red) at different timepoints. A‴,B‴,C‴,D‴ show the last round of immunolabelling of ribbon synapses (Ribeye-A, blue), amacrine and ganglion cells (HuC/D, magenta) and Müller glia (eGFP transgene, yellow) at different, crucial timepoints of retinal development (2, 2.5, 3 and 5 dpf) showing retinal progenitors in A–A‴. B–B‴ shows nascent IPL and Müller glia formation. In C–C‴ and D–D‴, IPL sublamination and Müller glia elaboration is evident. IPL, inner plexiform layer. All images representative of three experimental repeats. Scale bars: 25 µm (whole retina); 10 µm (magnified images).
	Fig. 8. IBEX on African clawed frog and turquoise killifish retina. (A–G) 5-month-old Xenopus laevis tadpole sagittal retinal sections labelled with HuC/D (B, yellow), PAX6 (C, purple), GS (D, magenta), GαO (E, green), cone opsin (F, cyan) and zpr-3 (G, red). HuC/D labelled cells within the GCL and amacrine cell sublayer, as seen in zebrafish, as well as photoreceptor inner segments. PAX6 labelled primarly cells within the GCL, potentially RGCs and/or displaced amacrine cells. Müller glia were labelled with GS. GαO labelled bipolar cell bodies and processes within the INL and IPL, respectively. L and M cone outer segments were co-labelled with CO and zpr-3. (H–Q) 8-week-old killifish retinal sections labelled with HuC/D (I, yellow), PAX6 (J, purple), GS (K, magenta), Calretinin (L, green), Zpr-1 (M, cyan), PCNA (N, red), PRPH2 (O, orange), Zrf-1 (P, light green) and Lcp1 (Q, lavender/light purple). Huc/D and PAX6 labelled cells within the GCL and amacrine cell sublayer, as seen in zebrafish and frogs. GS and zrf-1 labelled Müller glia. Calretinin labelled cells within the GCL and amacrine cell sublayer. Zpr-1 labelled entire double cone cells, and PRPH2 labelled photoreceptor outer segments. PCNA expression was observed in the CMZ and in the photoreceptor cell layer. Lcp1 was observed in the INL, close to the CMZ, and within the photoreceptor cell layer. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cell. All images representative of three experimental repeats Scale bars: 25 µm (Xenopus, A–G); 30 µm (killifish; H–Q).