Journal: International Journal of Molecular Sciences

Article Title: K Ca 1.1 K + Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

doi: 10.3390/ijms222413553

Figure Lengend Snippet: Comparison of K Ca 1.1 expression and activity between LNCaP cells cultured as 2D monolayers and 3D spheroids. ( A ): Phenotypic properties of LNCaP cells cultured in ultra-low attachment PrimeSurface 96U plates (upper panel: on day 0; lower panel: on day 7). Brightfield images were obtained with the Axio Observer Z1 microscope system (Carl Zeiss, Oberkochen, Germany). Bars show 500 μm. ( B ): Time course of the voltage-sensitive fluorescent dye imaging of K Ca 1.1 activator (1 μM NS1619)-induced hyperpolarizing responses in isolated cells from ‘2D’ monolayers and ‘3D’ spheroids of LNCaP. The fluorescent intensity of DiBAC 4 (3) before the application of NS1619 is expressed as 1.0. Images were measured every 5 s. ( C ): Summarized results of NS1619-induced hyperpolarizing responses in cells isolated from at least three different batches in each group. Cell numbers used in experiments are shown in parentheses. The values for fluorescent intensity were obtained by measuring the average for 1 min (12 images). ( D ): K Ca 1.1 protein expression in the lipid-raft-enriched protein lysates of both groups. Blots were probed with anti-K Ca 1.1 (approximately 100 kDa, upper panel) and anti-ACTB (43 kDa, lower panel) antibodies. ( E ): Summarized results were obtained as the optical density of K Ca 1.1 and ACTB band signals. After compensation for the optical density of the K Ca 1.1 protein band signal with that of the ACTB signal, the K Ca 1.1 signal in ‘2D’ was expressed as 1.0 ( n = 4 for each). ( F ): Real-time PCR examination of K Ca 1.1 in both groups ( n = 4 for each). Expression levels were shown as a ratio to ACTB. *: p < 0.05 vs. ‘2D’.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Comparison, Expressing, Activity Assay, Cell Culture, Spheroids, Brightfield, Microscopy, Imaging, Isolation, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: K Ca 1.1 K + Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

doi: 10.3390/ijms222413553

Figure Lengend Snippet: Effects of chemotherapy agents on the viability of 2D- and 3D-cultured LNCaP cells and the effects of a pretreatment with a K Ca 1.1 inhibitor on chemoresistance acquired by 3D-cultured LNCaP cells. ( A – D ): Effects of the treatment with 100 nM DTX, 100 nM PTX, 1 μM DOX, and 10 μM CIS for 48 h on the viability of ‘2D’- and ‘3D’-cultured LNCaP cells using the WST-1 assay ( n = 5 for each). Cell viability additing 0.1% dimethylsulfoxide, DMSO in DTX, PTX, and DOX and water in CIS instead of chemotherapy agents was expressed as 1.0. ( E – H ): Effects of the treatment with chemotherapy agents for 48 h on the viability of vehicle- and 10 μM PAX-pretreated (for 24 h), 3D-cultured LNCaP cells ( n = 5 for each). **: p < 0.01 vs. ‘2D’; ## : p < 0.01 vs. vehicle control.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Cell Culture, WST-1 Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: K Ca 1.1 K + Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

doi: 10.3390/ijms222413553

Figure Lengend Snippet: Effects of antiandrogens on the viability of 2D- and 3D-cultured LNCaP cells and the effects of a K Ca 1.1 inhibitor on the antiandrogen resistance acquired by 3D-cultured LNCaP cells. ( A , B ): Effects of the treatment with 10 μM BCT and 10 μM EZT for 48 h on the viability of ‘2D’- and ‘3D’-cultured LNCaP cells using the WST-1 assay ( n = 5 for each). Cell viability additing 0.1% DMSO instead of antiandrogens was expressed as 1.0. ( C , D ): Effects of the treatment with antiandrogens for 48 h on the viability of vehicle- and 10 μM PAX-pretreated (for 24 h), 3D-cultured LNCaP cells ( n = 5 for each). **: p < 0.01 vs. ‘2D’; ## : p < 0.01 vs. vehicle control.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Cell Culture, WST-1 Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: K Ca 1.1 K + Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

doi: 10.3390/ijms222413553

Figure Lengend Snippet: Expression of MRP transcripts in both 2D- and 3D-cultured LNCaP cells and the effects of K Ca 1.1 blockade with PAX on their expression in 3D-cultured LNCaP cells. (A–D): Real-time PCR examination of MRP1 ( A ), MRP3 ( B ), MRP4 ( C ), and MRP5 ( D ) in ‘2D’ monolayers and ‘3D’ spheroids of LNCaP cells ( n = 4 for each). ( E – H ): Real-time PCR examination of MRP transcripts in vehicle- and PAX-treated, 3D-cultured LNCaP cells ( n = 4 for each). Expression levels were shown as a ratio to ACTB. **: p < 0.01 vs. ‘2D’; ## : p < 0.01 vs. vehicle control.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Spheroids, Control

Journal: International Journal of Molecular Sciences

Article Title: K Ca 1.1 K + Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model

doi: 10.3390/ijms222413553

Figure Lengend Snippet: Regulation of K Ca 1.1 protein degradation through the FBXW7 ubiquitin E3 ligase in LNCaP cells. ( A , B ) Protein expression of FBXW7 in whole cell protein lysates of ‘2D’- and ‘3D’-cultured LNCaP cells. Blots were probed with anti-FBXW7 (approximately 70 kDa) and anti-ACTB (43 kDa) antibodies ( A ). Summarized results ( B ) were obtained as the optical densities of FBXW7 and ACTB band signals ( n = 4 for each). After compensation for the optical densities of the protein band signals with that of the ACTB signal, the optical density in ‘2D’ was expressed as 1.0 ( n = 4 for each). ( C , D ): Protein expression of K Ca 1.1 in control siRNA (si-Cont)- and FBXW7 siRNA (si-FBXW7)-transfected (for 72 h), 2D-cultured LNCaP cells. Blots were probed with anti-K Ca 1.1 (approximately 100 kDa) and anti-ACTB (43 kDa) antibodies ( C ). Summarized results ( D ) were obtained as the optical densities of K Ca 1.1 and ACTB band signals. The optical density in ‘si-Cont’ was expressed as 1.0 ( n = 4 for each). **: p < 0.01 vs. ‘2D’; # : p < 0.05 vs. ‘si-Cont’.

Article Snippet: Fixed and non-permeabilized cells were stained with an anti-K Ca 1.1 (extracellular) polyclonal antibody (rabbit) (APC-151, Alomone Labs) followed by an Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific), and then analyzed by flow cytometry (FACSCanto II, BD Biosciences, San Jose, CA, USA).

Techniques: Ubiquitin Proteomics, Expressing, Cell Culture, Control, Transfection, Western Blot

Journal: Frontiers in Immunology

Article Title: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event

doi: 10.3389/fimmu.2026.1951506

Figure Lengend Snippet: Fundoscopic examination of representative control (B6 and R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 ) and SAU (R161H +/- x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 ) retinas for infiltration of RFP + and RFP + GFP + cells. Age of mouse indicated. The same field of view for each mouse/time point was analyzed by brightfield fundoscopy and for RFP and GFP fluorescence. Arrows indicate areas of perivascular infiltration at 40 days of age in the R161H +/- x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 mouse (SAU score = 1.0).

Article Snippet: Primary antibodies against GFP (cat no. NB600-597, mouse anti-GFP, Novus Biologicals, Centennial, CO, USA) and RFP (cat no. NBP3-25634, rabbit anti-RFP, Novus Biologicals) were diluted 1:200 with DPBS and applied to the retinas and held at 4°C for at least 12 hours in dark conditions then washed 3 x 5 minutes with DPBS.

Techniques: Control, Brightfield, Fluorescence

Journal: Frontiers in Immunology

Article Title: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event

doi: 10.3389/fimmu.2026.1951506

Figure Lengend Snippet: Fluorescence microscopy of representative control (B6/J, Ccr2 RFP+/+ Cx3cr1 GFP+/+ , and R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 ) and SAU (R161H +/- x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 ) mouse retinas for RFP and GFP at 50 days of age. 40x magnification.

Article Snippet: Primary antibodies against GFP (cat no. NB600-597, mouse anti-GFP, Novus Biologicals, Centennial, CO, USA) and RFP (cat no. NBP3-25634, rabbit anti-RFP, Novus Biologicals) were diluted 1:200 with DPBS and applied to the retinas and held at 4°C for at least 12 hours in dark conditions then washed 3 x 5 minutes with DPBS.

Techniques: Fluorescence, Microscopy, Control

Journal: Frontiers in Immunology

Article Title: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event

doi: 10.3389/fimmu.2026.1951506

Figure Lengend Snippet: Flow cytometry distinguishes retinal MG from infiltrating macrophages. (A, B) Analysis of peripheral blood mononuclear cells for RFP and GFP expression showing the background levels of RFP and GFP fluorescence in B6/J mice (A) and the presence of RFP + , GFP + and RFP + GFP + cells in the circulation of Ccr2 RFP+/+ Cx3cr1 GFP+/+ mice (B) . The gating strategy for selecting CD45 + cells from B6/J mice is the same as shown for Ccr2 RFP+/+ Cx3cr1 GFP+/+ mice. (C) Representative analysis of a B6/J mouse showing background levels of RFP and GFP fluorescence in retinal CD45 + cells. Background fluorescence for GFP indicated as GFP neg cells. (D) Representative analysis of R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 retinas showing that a minimal number of all CD45 + cells (gate 1) express Ccr2 (RFP), and that a single population comprising most of the CD45 + cells and specifically the CD45 med cells (gate 2) express a high level of Cx3cr1 (GFP). Retinal CD45 + cells negative for GFP expression (GFP neg ) or with a low or intermediated level of GFP expression (GFP lo-med ) are indicated. (E) Representative analysis of R161H +/- x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 retinas with SAU showing a large increase in both CD45 med and CD45 hi cells with most of the RFP + cells found in the CD45 hi population as either RFP + GFP neg or RFP + GFP lo-med cells and that they are myeloid cells or lymphocytes. For all analyses; retinal MG indicated by red circles, flow cytometry done at 50 days of age, n = 4 for all groups of mice, and the mean percentage of cells in each quadrant for all mice analyzed is indicated.

Article Snippet: Primary antibodies against GFP (cat no. NB600-597, mouse anti-GFP, Novus Biologicals, Centennial, CO, USA) and RFP (cat no. NBP3-25634, rabbit anti-RFP, Novus Biologicals) were diluted 1:200 with DPBS and applied to the retinas and held at 4°C for at least 12 hours in dark conditions then washed 3 x 5 minutes with DPBS.

Techniques: Flow Cytometry, Analysis, Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event

doi: 10.3389/fimmu.2026.1951506

Figure Lengend Snippet: Analysis of R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 retinas for Ccr2 + (RFP + ) cells following ONC. (A) Representative fundoscopy of pre-ONC and day 10 post-ONC retinas. (B) Representative flow cytometry analysis of retinal CD45 med cells for RFP and GFP at day 10 post-ONC with mean percentage of CD45 med cells expressing GFP and RFP from all examined retinas indicated.

Article Snippet: Primary antibodies against GFP (cat no. NB600-597, mouse anti-GFP, Novus Biologicals, Centennial, CO, USA) and RFP (cat no. NBP3-25634, rabbit anti-RFP, Novus Biologicals) were diluted 1:200 with DPBS and applied to the retinas and held at 4°C for at least 12 hours in dark conditions then washed 3 x 5 minutes with DPBS.

Techniques: Analysis, Flow Cytometry, Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Integrated Histological and Transcriptomic Analyses Reveal Distinct Structural and Molecular Features of the Calipash and Muscle in the Chinese Soft-Shelled Turtle ( Pelodiscus sinensis )

doi: 10.3390/ani16182960

Figure Lengend Snippet: All the primers listed in this study.

Article Snippet: After blocking with 10% goat serum (Boster Biological Technology Co., Ltd., Wuhan, China) for 30 min, the sections were incubated with a rabbit anti-COL1A1 primary antibody (ABclonal Biotechnology Co., Ltd., Wuhan, China; 1:200 dilution) overnight at 4 °C.

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Integrated Histological and Transcriptomic Analyses Reveal Distinct Structural and Molecular Features of the Calipash and Muscle in the Chinese Soft-Shelled Turtle ( Pelodiscus sinensis )

doi: 10.3390/ani16182960

Figure Lengend Snippet: Quantitative analysis of collagen abundance in the calipash and muscle. ( A , B ) Representative Masson’s trichrome-stained sections utilized for collagen quantification in calipash ( A ) and muscle ( B ). ( A1 – A3 ) The first/second/third microscopic field in calipash; ( B1 – B3 ) The first/second/third microscopic field in muscle. The microscopic images are representative fields from the same individual and were used for visualization only. ( C ) Quantitative comparison of the collagen area fraction between the calipash and muscle tissues. Data are presented as mean ± SD ( n = 3) calculated from three independent microscopic fields. * p < 0.05 indicates significant differences.

Article Snippet: After blocking with 10% goat serum (Boster Biological Technology Co., Ltd., Wuhan, China) for 30 min, the sections were incubated with a rabbit anti-COL1A1 primary antibody (ABclonal Biotechnology Co., Ltd., Wuhan, China; 1:200 dilution) overnight at 4 °C.

Techniques: Analysis, Staining, Comparison

Journal: Animals : an Open Access Journal from MDPI

Article Title: Integrated Histological and Transcriptomic Analyses Reveal Distinct Structural and Molecular Features of the Calipash and Muscle in the Chinese Soft-Shelled Turtle ( Pelodiscus sinensis )

doi: 10.3390/ani16182960

Figure Lengend Snippet: Comparative Picro Sirius Red staining of collagen subtype composition and spatial organization. ( A1 – A4 ) Representative brightfield microscopy images of the calipash acquired at increasing magnification. Scale bars: 200 μm ( A1 ), 100 μm ( A2 ), 50 μm ( A3 ) and 20 μm ( A4 ). ( B1 – B4 ) Representative polarized light microscopy images of the calipash acquired at increasing magnification. Scale bars: 200 μm ( B1 ), 100 μm ( B2 ), 50 μm ( B3 ) and 20 μm ( B4 ). ( C1 – C4 ) Representative brightfield microscopy images of muscle acquired at increasing magnification. Scale bars: 200 μm ( C1 ), 100 μm ( C2 ), 50 μm ( C3 ) and 20 μm ( C4 ). ( D1 – D4 ) Representative polarized light microscopy images of muscle acquired at increasing magnification. Scale bars: 200 μm ( D1 ), 100 μm ( D2 ), 50 μm ( D3 ) and 20 μm ( D4 ). Yellow-orange birefringence indicates densely packed type I collagen, while green birefringence is indicative of type III collagen-rich regions. Scale bars are as indicated in the respective images.

Article Snippet: After blocking with 10% goat serum (Boster Biological Technology Co., Ltd., Wuhan, China) for 30 min, the sections were incubated with a rabbit anti-COL1A1 primary antibody (ABclonal Biotechnology Co., Ltd., Wuhan, China; 1:200 dilution) overnight at 4 °C.

Techniques: Staining, Brightfield, Microscopy, Light Microscopy

Journal: Animals : an Open Access Journal from MDPI

Article Title: Integrated Histological and Transcriptomic Analyses Reveal Distinct Structural and Molecular Features of the Calipash and Muscle in the Chinese Soft-Shelled Turtle ( Pelodiscus sinensis )

doi: 10.3390/ani16182960

Figure Lengend Snippet: Comparative immunofluorescence localization of COL1A1 in calipash ( A1 – A3 ) and muscle ( B1 – B3 ) tissues. ( A1 ) Representative immunofluorescence image showing the spatial distribution of COL1A1 (green) in calipash at low magnification; scale bar, 200 μm. ( A2 ) Higher-magnification image of a representative field of view in calipash; scale bar, 50 μm. ( A3 ) Higher-magnification image of a second, independent field of view in calipash; scale bar, 50 μm. ( B1 ) Spatial distribution of COL1A1 (red) in muscle tissue at low magnification; scale bar, 200 μm. ( B2 ) Higher-magnification image of a representative field of view in muscle; scale bar, 50 μm. ( B3 ) Higher-magnification image of a second, independent field of view in muscle; scale bar, 50 μm. Nuclei were counterstained with DAPI (blue). Scale bars are as indicated in the respective images.

Article Snippet: After blocking with 10% goat serum (Boster Biological Technology Co., Ltd., Wuhan, China) for 30 min, the sections were incubated with a rabbit anti-COL1A1 primary antibody (ABclonal Biotechnology Co., Ltd., Wuhan, China; 1:200 dilution) overnight at 4 °C.

Techniques: Immunofluorescence, Tissue

Journal: Animals : an Open Access Journal from MDPI

Article Title: Integrated Histological and Transcriptomic Analyses Reveal Distinct Structural and Molecular Features of the Calipash and Muscle in the Chinese Soft-Shelled Turtle ( Pelodiscus sinensis )

doi: 10.3390/ani16182960

Figure Lengend Snippet: Collagen gene expression profiling, transcriptional validation, and protein-level confirmation in calipash and muscle of P. sinensis . ( A ) Volcano plot of collagen-related DEGs. Each point represents a gene, with significantly upregulated (green) and downregulated (orange) genes in calipash defined by |log 2 fold change| ≥ 1 and FDR < 0.05. The x -axis represents log 2 fold change, and the y -axis represents log 10 adjusted p value. Collagen-related genes are highlighted, including col1a1 , col1a2 , col3a1 , col6a1 , col6a2 , col4a6 , col4a5 , col5a3 , col7a1 , col12a1 , col16a1 , and col17a1 . ( B ) Targeted qPCR validation of col1a1 and col1a2 expression in calipash and muscle. Data are presented as mean ± SD ( n = 3). Asterisks indicate significant differences between calipash and muscle ( p < 0.05). ( C ) Western blot analysis of COL1A1 protein in calipash and muscle. β-actin was used as the loading control.

Article Snippet: After blocking with 10% goat serum (Boster Biological Technology Co., Ltd., Wuhan, China) for 30 min, the sections were incubated with a rabbit anti-COL1A1 primary antibody (ABclonal Biotechnology Co., Ltd., Wuhan, China; 1:200 dilution) overnight at 4 °C.

Techniques: Gene Expression, Profiling, Biomarker Discovery, Expressing, Western Blot, Analysis, Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: Integrated Histological and Transcriptomic Analyses Reveal Distinct Structural and Molecular Features of the Calipash and Muscle in the Chinese Soft-Shelled Turtle ( Pelodiscus sinensis )

doi: 10.3390/ani16182960

Figure Lengend Snippet: DEGs related to collagen in the muscle and calipash of P. sinensis .

Article Snippet: After blocking with 10% goat serum (Boster Biological Technology Co., Ltd., Wuhan, China) for 30 min, the sections were incubated with a rabbit anti-COL1A1 primary antibody (ABclonal Biotechnology Co., Ltd., Wuhan, China; 1:200 dilution) overnight at 4 °C.

Techniques:

Journal: eLife

Article Title: An image segmentation method based on the spatial correlation coefficient of Local Moran’s I

doi: 10.7554/elife.89361

Figure Lengend Snippet: Figure 1. Calculation of Local Moran’s I in darkfield (a–j) and brightfield (k–t) light micrographs. (a) Large magnification fluorescent immunostaining for Kv4.2 in the ventral posteromedial (VPM) nucleus. (b) A 6 × 6 pixel segment of the image shown in a. The squares represent the pixels with grayscale values. (c) Normalized intensity values (zi). The purple framed area demonstrates the convolution step of calculations (see also methods steps 1–3). (d) A first-order convolution matrix (weight matrix, wij) was used for this calculation. (e) Weighted intensity values (wijzj). (f) Lagged gray values. (g) Local Moran’s I (Ii) values. (h) Pseudosignificance values for each pixel. (i) Masking the non-significant pixels (middle gray tone without numbers) resulted in two groups of pixels, where the intensity values significantly differ from a random distribution. (j) Considering the intensity values, the upper pixel group is interpreted as an object (red), the lower one as background (blue), and the gray field between them represent an area where the pixel intensities could come from a random distribution (interpreted as noise). (k–t) The same as a–j, but the calculations are shown on a bright field image. In this case, the low-intensity pixels representing the object are labeled with blue (t) high-intensity pixels (background) with red. Scale bars: (a) 5 μm, (k) 10 μm.

Article Snippet: DOI: https://doi.org/10.7554/eLife.89361 13 of 17 rabbit anti- Kv4.2 IgG (1:500; Alomone Labs, code APC- 023).

Techniques: Brightfield, Immunostaining, Labeling

Journal: eLife

Article Title: An image segmentation method based on the spatial correlation coefficient of Local Moran’s I

doi: 10.7554/elife.89361

Figure Lengend Snippet: Figure 3. Comparison of threshold-based, and Moran’s image selection on natural objects. (a) High-resolution confocal image (single optical plane) of Kv4.2 immunostaining in the VB complex of the mouse thalamus at three added noise levels (0, 80, and 160). (b) Majority projection of segmentation of the same images by expert human observers (n = 23, see methods). Pixels selected by the majority of observers are defined as ground truth. (c) Threshold-based segmentation with the best true positive rate [TPR]/false positive rate [FPR] values. (d) Moran’s based segmentation using matrix

Article Snippet: DOI: https://doi.org/10.7554/eLife.89361 13 of 17 rabbit anti- Kv4.2 IgG (1:500; Alomone Labs, code APC- 023).

Techniques: Comparison, Selection, Confocal, Immunostaining

Journal: eLife

Article Title: An image segmentation method based on the spatial correlation coefficient of Local Moran’s I

doi: 10.7554/elife.89361

Figure Lengend Snippet: Figure 4. High-resolution localization of Kv4.2 subunit in the ventral posteromedial (VPM) nucleus of the thalamus. (a) High-resolution light micrograph of a VPM neuron immunolabeled for the Kv4.2 subunit displaying intense immunopositive puncta (white arrows) along its proximal dendrites. (b) An electron micrograph showing Kv4.2 immunolabeling in the VPM using the preembedding immunogold technique. A proximal dendrite, identified by its large diameter (d), has more silver intensified gold particles (white arrowheads) than thin dendrites (d2, d3). Arrows, synapses; arrowheads, puncta

Article Snippet: DOI: https://doi.org/10.7554/eLife.89361 13 of 17 rabbit anti- Kv4.2 IgG (1:500; Alomone Labs, code APC- 023).

Techniques: Immunolabeling

Journal: eLife

Article Title: An image segmentation method based on the spatial correlation coefficient of Local Moran’s I

doi: 10.7554/elife.89361

Figure Lengend Snippet: Figure 5. Delineation of functionally relevant Kv4.2 ion channel clusters by Moran’s method. (a) High magnification confocal image of Kv4.2 immunostaining in the ventral posteromedial (VPM) nucleus of mouse thalamus. (b) Segmentation of Kv4.2 immunopositive objects by Moran’s (magenta outlines). (c) Segmentation of immunonegative areas by Moran’s (white outlines). (d) Confocal image of vGluT2 immunostaining (green channel) of the same section shown in (a). (e) Kv4.2 ion channel clusters which overlap with vGluT2-positive terminals (cyan outlines). (f) Kv4.2 ion channel clusters which do not overlap with vGluT2-positive terminals (yellow outlines). Blue arrows, Kv4.2 clusters overlapping with vGluT2 terminals; yellow arrows, Kv4.2 clusters non-overlapping with vGluT2 terminals (holds for a–f). (g) Histograms of the colocalizing and non-colacalizing Kv 4.2 immunopositive boutons pooled from three animals segmented by Moran’s method and binned by area (left) and intensity (right). Yellow bars, Kv4.2 objects non-overlapping with vGluT2; cyan bars, Kv4.2 objects overlapping with vGluT2. (h) Scatterplot of area vs staining intensity from three different animals. Yellow dots, Kv4.2 objects non-overlapping with vGluT2; cyan dots, Kv4.2 objects overlapping with vGluT2. (i, j) Silver particles (black arrowheads) indicating Kv4.2 immunoreactivity are localized on dendritic appendages (asterisks) and dendritic shafts (d) contacted by vGluT2-positive terminals (b, diaminobenzidine [DAB] positive) in the VPM. The plasma membrane of the same dendrites that are not contacted by vGluT2-positive terminals (white arrowheads) contains very few particles for the Kv4.2 subunit. Scale bars: (a–f) 5 μm, (i) 1 μm, (j) 0.5 μm.

Article Snippet: DOI: https://doi.org/10.7554/eLife.89361 13 of 17 rabbit anti- Kv4.2 IgG (1:500; Alomone Labs, code APC- 023).

Techniques: Confocal, Immunostaining, Staining, Clinical Proteomics, Membrane, Immunofluorescence, Transmission Electron Microscopy