filter set 43 he  (Carl Zeiss)

Journal: bioRxiv

Article Title: Two axolotl-adapted cell-ablation platforms reveal macrophage-dependent processes essential for spinal-cord and skeletal regeneration

doi: 10.64898/2026.02.10.705144

Figure Lengend Snippet: Validation of CD68:NTR 2.0-P2A-tdTomato transgenic line as a macrophage-specific reporter through multi-modal characterization combining transcriptomics, flow cytometry, cell morphology, and immunofluorescence. (A) Single-cell RNA sequencing analysis of regenerating limb tissue across a temporal time course showing cell-type-specific expression patterns. Heatmap demonstrates enrichment of CD68, CSF1R, and AIF1 in monocyte/macrophage clusters and CSF3R in granulocyte/neutrophil populations. UMAP showing clustering of 59128 cells over 8 timepoints. Li et al. dataset. (B) Flow cytometry gating strategy for isolation of CD68:tdTomato⁺ cells from 4 DPA regenerating limbs. tdTomato+ cells were around 30% of the dissociated total population. (C) Representative image of FACS-sorted CD68:tdTomato⁺ cytospin preparations stained with DAPI demonstrates characteristic monocyte/macrophage morphology (magenta) including kidney-shaped nuclei (blue) (N=3 independent sorts). Scale Bar: 20 µm. (D) Quantitative RT-PCR analysis of FACS-sorted CD68:tdTomato⁺ versus tdTomato⁻ populations. Bar graphs show enrichment of macrophage markers (CD68, CSF1R) in tdTomato⁺ cells and enrichment of B-cell (IGHM) and neutrophil (CSF3R) markers in tdTomato⁻ fractions. Cells were isolated by FACS from three transgenic animals and pooled per condition prior to RNA extraction to obtain sufficient material (N=3 animals, ∼1-year-old, ∼12 cm length). Each sample was analyzed in triplicate technical qPCR reactions; dots represent individual technical replicates and bars represent the mean +/- SD. Gene expression is shown as fold change, calculated using the ΔΔCt method with normalization to RBL27 and Beta-actin reference genes. Data are shown from a representative experiment; the experiment was repeated independently with similar results. (E) Confocal maximum intensity projections of 7 DPA CD68:tdTomato transgenic limb section shows coexpression of tdTomato (Magenta) with the canonical macrophage marker Iba1(AIF1; in green), shown with white arrows. Merged image with DAPI (blue) confirms nuclear localization. Tissue-resident macrophages found in the wound epithelium (cyan inset) and circulating monocytes recruited to the blastema (orange inset) are both shown to be CD68/Iba1 + . Scale Bar: 200 µm. Inset Scale Bar: 20 µm. DPA=Days post-amputation. Antibodies: Rabbit anti-RFP (1:200, Rockland cat. 600-401-379); Chicken anti-Iba1 (1:50, Antibodies Inc. cat. IBA1-0020).

Article Snippet: DAPI, tdTomato, NucView 488, and CF640R fluorescence was excited and collected by using Zeiss DAPI filter cube set 49 (ref: 488049-9901-000), Zeiss EGFP filter cube set 38 HE (ref: 89038-9901-000), Zeiss mCherry filter cube set 63 HE (ref: 489063-0000-000), and Zeiss Cy5 filter cube set 50 (ref: 488050-9901-000) respectively.

Techniques: Biomarker Discovery, Transgenic Assay, Transcriptomics, Flow Cytometry, Immunofluorescence, Single Cell, RNA Sequencing, Expressing, Isolation, Staining, Quantitative RT-PCR, RNA Extraction, Gene Expression, Marker

Journal: bioRxiv

Article Title: Two axolotl-adapted cell-ablation platforms reveal macrophage-dependent processes essential for spinal-cord and skeletal regeneration

doi: 10.64898/2026.02.10.705144

Figure Lengend Snippet: Tail amputations were performed on germline F1 CD68:NTR 2.0-P2A-tdTomato (CD68:NTR 2.0) or CD68:mScarlet-IRES-ihCasp9 (CD68:IRES-ihCasp9) transgenic larvae alongside age and size matched wildtype d/d then subjected to repeated inducible drug treatments to maintain macrophage depletion throughout the regenerative process. (A) Experimental timeline of NTR 2.0 macrophage ablation and non-sensitized d/d toxicity evaluation shows chronic (14 day – long red arrow) versus four acute (18 hours every 3 days - red solid blocks) doses of MTZ administered through waterborne immersion (WB) with imaging schedule. Imaging timepoints shown with microscope icon and time relative to amputation. (B) Experimental timeline of ihCasp9 macrophage ablation and non-sensitized d/d toxicity evaluation shows 10 mg/kg B/B intraperitoneal injection ( i.p. ) regimen (every 2 days for 23 days) with imagining schedule. Timepoints with i.p. drug delivery of B/B shown in magenta. Imaging timepoints shown with microscope icon and time relative to amputation. (C, D) Representative extended depth of focus projections show CD68:NTR 2.0 transgenic tails at 2 and 14 DPA: (C) 1% DMSO vehicle control, (D) chronic 1 mM MTZ-treated (N=5 animals per treatment, 2-month-old, 2-3 cm length). Amputation plane indicated by white dotted line and cyan boxed regions mark the inset location. (C’, D’) Insets show decreased CD68:NTR 2.0 recruitment and rounded macrophage morphology within the regenerative zone when treated with MTZ relative to DMSO control. Thin white dotted lines outline the spinal cord (dorsal) and cartilage (ventral). Scale Bar: 500 µm. Inset Scale Bar: 100 µm. (E, F) Representative extended depth of focus projections show CD68:IRES-ihCasp9 transgenic tails at 2 and 14 DPA: (E) vehicle control, (F) 10 mg/kg B/B-treated (N=3-5 animals per treatment, 2-month-old, 2-3 cm length). Amputation plane indicated by white dotted line and cyan boxed regions mark the inset location. (E’, F’) Insets show fragmented CD68:IRES-ihCasp9 debris with complete inhibition of both cartilage and spinal cord regeneration relative to the vehicle control. Thin white dotted lines outline the spinal cord (dorsal) and cartilage (ventral). Scale Bar: 500 µm. Inset Scale Bar: 100 µm. (G) Quantitative morphometric analysis of tail regenerates in CD68:NTR 2.0 transgenics (solid lines) and non-sensitized d/d controls (dashed lines) treated with MTZ. Measurements of cartilage length, spinal cord length, and epithelial surface area that extend beyond the amputation plane were collected at 2, 7, 14, and 21 DPA in Fiji (N=5 animals per treatment, 2-month-old, 2-3 cm length). (H) Quantitative morphometric analysis of tail regenerates in CD68:IRES-ihCasp9 transgenics (solid lines) and non-sensitized d/d controls (dashed lines) shows cartilage length, spinal cord length, and epithelial surface area relative to amputation plane measured at 2, 7, 14, and 21 DPA in Fiji (N=3-5 animals per treatment, 2-month-old, 2-3 cm length). (I) Representative maximum intensity projections of Victoria blue stained CD68:IRES-ihCasp9 transgenic tails at 21 DPA demonstrate regenerative failure following amputation of the cartilaginous rod in B/B-treated animals compared to the vehicle-treated controls (N=3 animals per treatment, 2-month-old, 2-3 cm length). Amputation plane indicated by white dotted line. Scale Bar: 100 µm. (J) Representative confocal maximum intensity projections of vehicle-versus B/B-treated CD68:IRES-ihCasp9 transgenic tails stained with DAPI (blue), βIII-tubulin + axons (green), GFAP⁺ radial glial cells (magenta), and collagen I (yellow) at 21 DPA (N=3 animals per treatment, 2-month-old, 2-3 cm length). Amputation plane indicated by white dotted line. Scale Bar: 100 µm. Error bars represent mean ± SD. Statistical significance of inducible drug treatments relative to the respective vehicle controls at each regenerative timepoint was determined by two-way ANOVA with Tukey’s multiple comparisons test (* p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001). MTZ=Metronidazole (NTR 2.0 prodrug), B/B=AP20187 (ihCasp9 homodimerizer), DPA=Days post-amputation.

Article Snippet: DAPI, tdTomato, NucView 488, and CF640R fluorescence was excited and collected by using Zeiss DAPI filter cube set 49 (ref: 488049-9901-000), Zeiss EGFP filter cube set 38 HE (ref: 89038-9901-000), Zeiss mCherry filter cube set 63 HE (ref: 489063-0000-000), and Zeiss Cy5 filter cube set 50 (ref: 488050-9901-000) respectively.

Techniques: Transgenic Assay, Imaging, Microscopy, Injection, Control, Inhibition, Staining

Journal: bioRxiv

Article Title: Two axolotl-adapted cell-ablation platforms reveal macrophage-dependent processes essential for spinal-cord and skeletal regeneration

doi: 10.64898/2026.02.10.705144

Figure Lengend Snippet: Whole-mount immunofluorescence analysis of CD68:mScarlet-IRES-ihCasp9 transgenic tails at 21 days post-amputation reveals profound disruption of nervous system regeneration following sustained macrophage ablation with 10 mg/kg AP20187 (B/B; ihCasp9 homodimerizer) administered via intraperitoneal injection. (A, B) Representative confocal maximum intensity projections of whole-mounted CD68:mScarlet-IRES-ihCasp9 tails at 21 DPA (vehicle-versus B/B-treated) stained for acetylated tubulin⁺ axons (green), HNK1⁺ neural crest-derived Schwann cells (magenta), and DAPI nuclear counterstain (blue)(N=3 animals per treatment, 2-month-old, 2-3 cm length). Amputation plane indicated by white dotted line. Cyan boxed regions indicate the dorsal inset location while orange boxed regions indicate the midline inset location. Scale Bar: 200 µm (A’, B’) High-magnification insets (cyan boxes) of dorsal regions at the amputation plane. Normal regenerative axon outgrowth beyond amputation plane shown with white arrowhead (acetylated tubulin:green) absent in Mϕ-depleted (B/B-treated tails). Normal HNK1+ (Magenta) neural-crest-derived Schwann cell myelinated axons in peripheral nerves present in control (white arrows) missing in Mϕ-depleted (B/B-treated tails) showing displacement of HNK1+ cells away from the midline. Scale Bar: 100 µm. (A’’, B’’) High-magnification insets (orange boxes) of midline regions show spinal cord axonal architecture. Large hollow arrowhead highlights axonal degeneration proximal to amputation plane in B/B-treated animals (B’’) , contrasting with robust axonal extension beyond the amputation plane in vehicle controls (A’’) . Scale Bar: 100 µm. CD68:tdTomato + cells stained with anti-tdTomato antibody (magenta). Macrophages stained with anti-Iba1/AIF1 (green). Axons stained with Acetylated tubulin (yellow).

Article Snippet: DAPI, tdTomato, NucView 488, and CF640R fluorescence was excited and collected by using Zeiss DAPI filter cube set 49 (ref: 488049-9901-000), Zeiss EGFP filter cube set 38 HE (ref: 89038-9901-000), Zeiss mCherry filter cube set 63 HE (ref: 489063-0000-000), and Zeiss Cy5 filter cube set 50 (ref: 488050-9901-000) respectively.

Techniques: Immunofluorescence, Transgenic Assay, Disruption, Injection, Staining, Derivative Assay, Control