Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: Illustration of MACSima™ Imaging Cyclic Staining (MICS) principle MICS technology was applied (Step 46). (0) Image acquisition of 3D-IF staining in autofluorescence channel, followed by Photobleaching. (2–4) Multi-cyclic imaging: Rounds of 2D-IF staining with FITC, PE and APC coupled antibody fluorochrome-conjugate, image acquisition of respective FITC, PE and APC-channels and signal erasure by photobleaching.

Article Snippet: Figure 7 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”).

Techniques: Imaging, Staining

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: Figure 7 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”).

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Human OvCa) (A) Imaris 3D surface rendering of autofluorescence (cyan) and CD326 positive cells (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) with target plane in yellow. (C) Light sheet guided target plane selection representing CD326 positive cell (purple), CD45 positive cells (red), and CD3 positive cells (green). (D) DAPI overview image of selected tissue slice for 2D MACSima™ imaging. (E) Magnified merged six color multiparameter MICS image with CD45 (green), CD326 (cyan), FOLR1 (purple), Collagen III (red), Collagen IV (red), and CD31 (yellow). (F–L) Single staining MICS images (white) of DAPI (F), CD45 (G), CD326 (H), FOLR1 (I), Collagen III (J), Collagen IV (K), and CD31 (L) (gray) (see “Antibodies”). Scale bars: (A–F) 1 mm; (E) 250 μm; (F–L) 500 μm.

Article Snippet: Figure 7 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”).

Techniques: Imaging, Comparison, Selection, Staining

Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: Target sectioning – Transition from 3D light sheet to 2D multi-cyclic imaging Sample rehydration in descending ethanol series and PBS (100%, 90%, 70%, 50%, 30%, PBS and PBS) and cryoprotection with 30% sucrose solution (Step 30). Angle correction through angled agarose blocks and embedding in optimal cutting compound (Step 33). Snap freezing with isopentane and liquid nitrogen (Step 34). Iterative feedback loop to verify simulated cutting path with cryosectioning in 10 μm increments (Step 35), navigation through anatomical landmarks and 3D-IF fluorescence signature with stereo/fluorescence microscopy (Step 36) and comparison of optical physical sections versus theoretical optical sections (Step 37). This process is repeated until target plane is reached (feedback loop, Step 35–37). Scale bars: (Verification) 500 µm.

Article Snippet: Figure 7 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”).

Techniques: Imaging, Fluorescence, Microscopy, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: A switch from α5β1 to αvβ3 integrin activity contributes to the development of a profibrotic mesenchymal phenotype in trabecular meshwork cells

doi: 10.3389/fcell.2025.1730542

Figure Lengend Snippet: Activity of αvβ3 integrin affects the expression of VIM , SNAI2 , and TWIST1 mRNA levels. (A) Flow cytometry showed that TM cells (Old α5-) derived from 74 to 77-year-old donor eyes expressed lower levels of α5 integrin compared to TM cells derived from young (young α5+) and other old donor eyes (old α5+). The designation α5+ refers to the fact that a large percentage of cells express the α5 integrin subunit while α5- refers to the fact that most of these cells do not express the α5 integrin subunit. (B) Despite differences in the levels of the α5 integrin subunit, all three populations of cells expressed similar levels of β3 integrin. (C) More old a5- TM cells (N74 and N77) expressed active αvβ3 integrin on the cell surface compared to young and old TM cells expressing α5β1 integrins; * p < 0.02. N = 10,000 cells per condition. N = 7 young α5+ biological replicates (ages 17–36), N = 5 old α5+ biological replicates expressing α5β1 integrin (ages 55–75). N = 2 old α5- biological replicates (ages 74–77). Cells were labeled with P1D6 (α5β1 integrin), LM609 (total αvβ3 integrin), or LIBS2 (active αvβ3 integrin) mAbs. (D,E) RT-qPCR showed that Old α5- TM cells expressed significantly higher levels ( p < 0.04) of mRNA for the EndMT markers VIM and SNAI2 compared to cells isolated from young α5+ donor eyes (N25 and N35). (F) TWIST1 mRNA levels were also higher in the old α5 integrin negative cells but the levels were not statistically significant ( p < 0.08). (G–I) Knockdown of β3 integrin using shRNA in the old N77 cells (Old α5-) that contained elevated levels of active αvβ3 integrin and low levels of α5β1 integrin mRNA had statistically reduced levels of VIM ( p < 0.0004) and TWIST1 ( p < 0.01) mRNA. Levels of SNAI2 mRNA levels were also reduced, but not statistically ( p < 0.07). All experiments were done in triplicates and repeated twice.

Article Snippet: Briefly, TM cells were lifted with Cell Dissociation Solution Non-enzymatic (Sigma-Aldrich Corp.), blocked for 30 min on ice with 1% BSA in PBS and labeled for 1 h on ice with 10 μg/mL α5 (P1D6, ThermoFisher Scientific, #12-4900-42, RRID:AB_10717080), total αvβ3 (LM609, Sigma-Millipore, mAb 1976, RRID:AB_2296419), and active αvβ3 (LIBS2, Millipore Sigma, MABT27, RRID:AB_10806476) integrin antibodies in 1% BSA/PBS.

Techniques: Activity Assay, Expressing, Flow Cytometry, Derivative Assay, Labeling, Quantitative RT-PCR, Isolation, Knockdown, shRNA