Zen blue software v2, by Zeiss - Product details

1

Spatial Transcriptomics with Visium Platform

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When using the Tissue Optimization workflow, a fluorescent image (mRFP filter, Excitation: 559/85 Emission: 600/90) of the cDNA footprint was taken. The whole slide was recorded using a Plan-Apochromat 10×/0.45 M27 objective and the tiling function of the Axio-Imager.M2 microscope (Zeiss). The final file included each of the eight capture areas, 8 × 8 mm (the fiducial frame and the capture area); with the microscope set to ∼1–2 mm beyond the fiducial frame for optimal image alignment. The capture resolution was 0.454 μm/pixel. Raw images were stitched together using Zen blue software v2.5 (Zeiss). When following the Gene Expression workflow, libraries from the different capture areas were synthesized as described in 10x Genomics User guide (PN-1000190, CG000239_VisiumSpatialGeneExpression_UserGuide_RevD).
Visium libraries were sequenced on an Illumina NextSeq 550 platform according to the 10× Genomics Visium manufacturer's instructions (NextSeq 500/550 High Output kit v2.5 (150 cycles) 20024907, CG000239_VisiumSpatialGeneExpression_UserGuide_RevD), targeting 100 million reads using dual indexing kit TT set A (PN-1000215, 10x Genomics).

Peirats-Llobet M., Yi C., Liew L.C., Berkowitz O., Narsai R., Lewsey M.G, & Whelan J. (2023). Spatially resolved transcriptomic analysis of the germinating barley grain. Nucleic Acids Research, 51(15), 7798-7819.

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Safranin O Staining for Visium Imaging

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The slides were dried on a metal plate (PN-1000317, 10x Genomics) in the thermocycler at 37°C for 1 min, fixed with chilled methanol at –20°C for 30 min, stained with 0.1% (w/v) Safranin O (Sigma-Aldrich, cat no. S8884-25G) in 50% (vol/vol) Ethanol with 2 U/μl RNase inhibitor at room temperature (RT) for 5 min and washed in increasing concentrations of EtOH until the discarded liquid was clear. After drying at 37°C for 1 min, the slides were mounted in 85% (v/v) glycerol + 2 U/μl RNase inhibitor, covered with a coverslip and images of sections were taken using Axio-Imager.M2 microscope (Zeiss). TO slides were captured in on image while GE slide capture areas were imaged individually. Raw images were stitched together using Zen blue software v2.5 (Zeiss). Prior to imaging, the microscope settings were validated using the Visium Imaging Test Slide (PN-2000235). The optimised settings were saved into the TO macro for following experiments. After imaging, the coverslip was removed immediately, and the slide immersed at 45° angle in 3× SSC buffer and washed once with 3× SSC buffer before air drying.

Peirats-Llobet M., Yi C., Liew L.C., Berkowitz O., Narsai R., Lewsey M.G, & Whelan J. (2023). Spatially resolved transcriptomic analysis of the germinating barley grain. Nucleic Acids Research, 51(15), 7798-7819.

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3

Immunohistochemical analysis of intestinal cell markers

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5 μm paraffin sections were deparaffinized by three times immersion in xylene (5 min each time) and rehydrated by immersion in a series of graded ethanol dilutions 100%, 90%, and 70% for 5 min each. Epitope retrieval was performed by preheating the sections 5 min at full power microwave (900 W) in 10 mM sodium citrate buffer pH 6.5 until boiling, followed by 10 min at a sub-boiling temperature (600 W). After cooling down for 20 min, the sections were washed in PBS and blocked with 1% BSA/PBS for 1 h at RT in humid chamber. Sections were stained with primary antibodies: anti-Olfm4 (Cell Signaling, D6Y5A, #39141), anti-Muc2 (abcam, ab90007), and anti-Chga (abcam, ab15160) in 1% BSA/PBS for 16 h at 4 degree in humid chamber. This was followed by washing in PBST (0.1% Tween 20, 3 × 5 min) and subsequent incubation for 30 min with secondary anti-rabbit IgG conjugated with AF488. All the antibodies were used at concentration of 1:250. The slides were washed in T-PBS (0.1% Tween 20, 3 × 5 min) and mounted with mounting medium including DAPI. Images of stained sections were acquired using Axio Imager from Zeiss and analyzed by the ZEN blue software v2 (Zeiss). For further image analysis, the graphics tools for counting and measuring the ZEN software were used. Details on antibodies can be found in Supplementary Table S2.

Omrani O., Krepelova A., Rasa S.M., Sirvinskas D., Lu J., Annunziata F., Garside G., Bajwa S., Reinhardt S., Adam L., Käppel S., Ducano N., Donna D., Ori A., Oliviero S., Rudolph K.L, & Neri F. (2023). IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration. Nature Communications, 14, 6109.

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High-Resolution Tissue Imaging Protocol

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Images of sections stained with either Alcian Blue or Hematoxylin and Eosin were acquired using Axio Imager or Axio Scan imaging systems (both from Zeiss) and the ZEN blue software v2 (Zeiss). Images of sections were acquired as individual tiles using 10x objectives after selection of 9–15 focus points per section. Tiles were automatically stitched together by ZEN. For further image analysis, the graphics tools for counting and measuring of the ZEN software were used.

Gebert N., Cheng C.W., Kirkpatrick J.M., Fraia D.D., Yun J., Schädel P., Pace S., Garside G.B., Werz O., Rudolph K.L., Jasper H., Yilmaz Ö.H, & Ori A. (2020). Region-Specific Proteome Changes of the Intestinal Epithelium during Aging and Dietary Restriction. Cell reports, 31(4), 107565.

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5

High-Resolution Tissue Imaging Protocol

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Images of sections stained with either Alcian Blue or Hematoxylin and Eosin were acquired using Axio Imager or Axio Scan imaging systems (both from Zeiss) and the ZEN blue software v2 (Zeiss). Images of sections were acquired as individual tiles using 10x objectives after selection of 9–15 focus points per section. Tiles were automatically stitched together by ZEN. For further image analysis, the graphics tools for counting and measuring of the ZEN software were used.

Gebert N., Cheng C.W., Kirkpatrick J.M., Fraia D.D., Yun J., Schädel P., Pace S., Garside G.B., Werz O., Rudolph K.L., Jasper H., Yilmaz Ö.H, & Ori A. (2020). Region-Specific Proteome Changes of the Intestinal Epithelium during Aging and Dietary Restriction. Cell reports, 31(4), 107565.

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6

Immunohistochemical analysis of intestinal cell markers

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5 μm paraffin sections were deparaffinized by three times immersion in xylene (5 min each time) and rehydrated by immersion in a series of graded ethanol dilutions 100%, 90%, and 70% for 5 min each. Epitope retrieval was performed by preheating the sections 5 min at full power microwave (900 W) in 10 mM sodium citrate buffer pH 6.5 until boiling, followed by 10 min at a sub-boiling temperature (600 W). After cooling down for 20 min, the sections were washed in PBS and blocked with 1% BSA/PBS for 1 h at RT in humid chamber. Sections were stained with primary antibodies: anti-Olfm4 (Cell Signaling, D6Y5A, #39141), anti-Muc2 (abcam, ab90007), and anti-Chga (abcam, ab15160) in 1% BSA/PBS for 16 h at 4 degree in humid chamber. This was followed by washing in PBST (0.1% Tween 20, 3 × 5 min) and subsequent incubation for 30 min with secondary anti-rabbit IgG conjugated with AF488. All the antibodies were used at concentration of 1:250. The slides were washed in T-PBS (0.1% Tween 20, 3 × 5 min) and mounted with mounting medium including DAPI. Images of stained sections were acquired using Axio Imager from Zeiss and analyzed by the ZEN blue software v2 (Zeiss). For further image analysis, the graphics tools for counting and measuring the ZEN software were used. Details on antibodies can be found in Supplementary Table S2.

Omrani O., Krepelova A., Rasa S.M., Sirvinskas D., Lu J., Annunziata F., Garside G., Bajwa S., Reinhardt S., Adam L., Käppel S., Ducano N., Donna D., Ori A., Oliviero S., Rudolph K.L, & Neri F. (2023). IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration. Nature Communications, 14, 6109.

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7

Immunohistochemical analysis of intestinal cell markers

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5 μm paraffin sections were deparaffinized by three times immersion in xylene (5 min each time) and rehydrated by immersion in a series of graded ethanol dilutions 100%, 90%, and 70% for 5 min each. Epitope retrieval was performed by preheating the sections 5 min at full power microwave (900 W) in 10 mM sodium citrate buffer pH 6.5 until boiling, followed by 10 min at a sub-boiling temperature (600 W). After cooling down for 20 min, the sections were washed in PBS and blocked with 1% BSA/PBS for 1 h at RT in humid chamber. Sections were stained with primary antibodies: anti-Olfm4 (Cell Signaling, D6Y5A, #39141), anti-Muc2 (abcam, ab90007), and anti-Chga (abcam, ab15160) in 1% BSA/PBS for 16 h at 4 degree in humid chamber. This was followed by washing in PBST (0.1% Tween 20, 3 × 5 min) and subsequent incubation for 30 min with secondary anti-rabbit IgG conjugated with AF488. All the antibodies were used at concentration of 1:250. The slides were washed in T-PBS (0.1% Tween 20, 3 × 5 min) and mounted with mounting medium including DAPI. Images of stained sections were acquired using Axio Imager from Zeiss and analyzed by the ZEN blue software v2 (Zeiss). For further image analysis, the graphics tools for counting and measuring the ZEN software were used. Details on antibodies can be found in Supplementary Table S2.

Omrani O., Krepelova A., Rasa S.M., Sirvinskas D., Lu J., Annunziata F., Garside G., Bajwa S., Reinhardt S., Adam L., Käppel S., Ducano N., Donna D., Ori A., Oliviero S., Rudolph K.L, & Neri F. (2023). IFNγ-Stat1 axis drives aging-associated loss of intestinal tissue homeostasis and regeneration. Nature Communications, 14, 6109.

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8

Microscopy Imaging of Paralyzed Worms

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For microscopy, animals were either picked from mixed staged plates or from bleach synchronized populations. Worms were paralyzed in 20 mM sodium azide and mounted on a pad of 4 % Noble Agar in 50 % M9 buffer supplemented with 20 mM sodium azide. They were examined with a Zeiss AxioImager equipped with Colibri LED illumination and an Apotome. Generally, image stacks were recorded with 0.5 μm distance between slices. If the fluorescent signal was too dim for Apotome imaging, the deconvolution algorithm implemented in the Zeiss ZenBlue software v. 2.6 was used (“good/fast iterative” setting) to improve image quality. Some DIC and fluorescent images were generated using a Nikon Ti-S compound inverted microscope equipped with a Hamamatsu C11440–10C Flash 2.8 camera. The NIS Elements Version 4.2 software was used for image acquisition, and digital images were processed using the Adobe Photoshop Elements software (Adobe Systems, San Jose, CA).

Kiontke K., Herrera R.A., Mason D.A., Woronik A., Vernooy S., Patel Y, & Fitch D.H. (2024). Tissue-specific RNA-seq defines genes governing male tail tip morphogenesis in C. elegans. bioRxiv.

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9

Paralysis and Imaging of Worms

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For microscopy, worms were paralyzed in 20 mM sodium azide and mounted on a pad of 4 % Noble Agar in 50 % M9 buffer supplemented with 20 mM sodium azide. They were examined with a Zeiss AxioImager equipped with Colibri LED illumination and an Apotome. Generally, image stacks were recorded with 0.5 μm distance between slices. If the fluorescent signal was too dim for Apotome imaging, the deconvolution algorithm implemented in the Zeiss ZenBlue software (v. 2.6) was used (“good/fast iterative” setting) to improve image quality. In some cases, several images in a stack were combined in a Z-projection using Zen Blue. 3D reconstructions were also made with Zen Blue.

Kiontke K., Fernandez P., Woronik A, & Fitch D.H. (2024). Morphologically defined substages of tail morphogenesis in C. elegans males. bioRxiv.

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Zen blue software v2

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9 protocols using zen blue software v2

Spatial Transcriptomics with Visium Platform

Safranin O Staining for Visium Imaging

Immunohistochemical analysis of intestinal cell markers

High-Resolution Tissue Imaging Protocol

High-Resolution Tissue Imaging Protocol

Immunohistochemical analysis of intestinal cell markers

Immunohistochemical analysis of intestinal cell markers

Microscopy Imaging of Paralyzed Worms

Paralysis and Imaging of Worms

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