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clip170  (Addgene inc)


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    Addgene inc clip170
    (a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, <t>CLIP170,</t> or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).
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    Images

    1) Product Images from "A single-component optogenetic toolkit for programmable control of microtubule"

    Article Title: A single-component optogenetic toolkit for programmable control of microtubule

    Journal: bioRxiv

    doi: 10.1101/2025.10.31.685931

    (a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, CLIP170, or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).
    Figure Legend Snippet: (a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, CLIP170, or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).

    Techniques Used: Binding Assay, Derivative Assay, Construct, Variant Assay, Activity Assay, Expressing, Fluorescence, Control, Labeling, Staining, Immunostaining, Live Cell Imaging

    Related Articles

    Plasmid Preparation:

    Article Title: A single-component optogenetic toolkit for programmable control of microtubule
    Article Snippet: Tubulin Tracker (Deep Red, Cat No. T34077) and Goat anti-mouse IgG highly cross-adsorbed secondary antibodies (conjugated with Alexa Fluor 488 or 555; Cat No. #A-11001, and A-21422) were purchased from Thermo Fisher Scientific. .. The plasmid templates for EB1 (#17234), CLIP170 (#54044), CAMSAP1 (# 59036),CAMSAP2 (#59037), KIF5A (#166954), and spastin (#134461) were purchased from Addgene. .. To generate OptoMT, we firstly amplified the PHR domain (residues 1-498) of Arabidopsis thaliana CRY2 (Addgene, #70159) by standard PCR and then inserted the fragment into modified pmCherry-C1 and pEGFP-C1 vectors (Clontech), followed by the insertion of multiple MT binding domains, derived from EB1, CLIP170, KIF5A, or CAMSAP1 at the BspEI and EcoRI/BamHI sties, as well as a 3x(SGGGGG) flexible linker between CRY2 and the MT binding domain.

    Article Title: Genetically Encoded Microtubule Binders for Single-Cell Interrogation of Cytoskeleton Dynamics and Protein Activity.
    Article Snippet: Goat antimouse IgG highly cross-adsorbed secondary antibodies (conjugated with Alexa Fluor 488, Cat No. #A-11001) were purchased from Thermo Fisher Scientific. .. Plasmid Construction. pGFP-EB1 (Addgene no. 17234), CLIP170 (no. 54044), and MDM2-YFP (no. 53962) were purchased from Addgene. .. The oligomerization domains from the p53 family and EB1-binding SxIP motif (DST5474−5485) were directly synthesized as DNA oligos by Integrated DNA Technologies.

    Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis
    Article Snippet: .. The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table . .. PCR fragments of IQGAP-DBR (1500–1657aa) were cloned into pDEST15 destination vector (Life Technologies) using In-Fusion Cloning Kit (Clontech) to generate GST fusion proteins.

    Polymerase Chain Reaction:

    Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis
    Article Snippet: .. The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table . .. PCR fragments of IQGAP-DBR (1500–1657aa) were cloned into pDEST15 destination vector (Life Technologies) using In-Fusion Cloning Kit (Clontech) to generate GST fusion proteins.

    Generated:

    Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis
    Article Snippet: .. The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table . .. PCR fragments of IQGAP-DBR (1500–1657aa) were cloned into pDEST15 destination vector (Life Technologies) using In-Fusion Cloning Kit (Clontech) to generate GST fusion proteins.

    Analogues:

    Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis
    Article Snippet: .. The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table . .. PCR fragments of IQGAP-DBR (1500–1657aa) were cloned into pDEST15 destination vector (Life Technologies) using In-Fusion Cloning Kit (Clontech) to generate GST fusion proteins.

    Clone Assay:

    Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis
    Article Snippet: .. The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table . .. PCR fragments of IQGAP-DBR (1500–1657aa) were cloned into pDEST15 destination vector (Life Technologies) using In-Fusion Cloning Kit (Clontech) to generate GST fusion proteins.

    Amplification:

    Article Title: Inhibition of polar actin assembly by astral microtubules is required for cytokinesis
    Article Snippet: .. The PCR fragments were mixed and reamplified using 5’ and 3’ oligos of DIAPH1. cDNAs of DIAPH1-CT were generated and fused to phospholipase Cδ1-PH (PLCδ1-PH) using analogues strategy to generated cDNAs of PLCδ1-PH-DIAPH1-CT. Alternatively, PCR fragments of DIAPH1-CT were cloned using the TOPO Gateway system (Life Technologies) being first cloned into the entry plasmid vector pCR8/GW/TOPO, then moved into the destination vectors pKM596 (Addgene plasmid 8837) to generate MBP fusion proteins. cDNAs of full-length human IQGAP1 (1–1657aa), CLIP170 (1–1320aa), or fragments of IQGAP1-DBR (1500–1657aa), CLIP170-NT (1–350aa), CLIP170-CT (500–1320aa) were amplified using the i-Max II DNA polymerase (Froggalab) using oligonucleotide primers listed in Supplementary Table . .. PCR fragments of IQGAP-DBR (1500–1657aa) were cloned into pDEST15 destination vector (Life Technologies) using In-Fusion Cloning Kit (Clontech) to generate GST fusion proteins.



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    (a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, <t>CLIP170,</t> or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).
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    (a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, <t>CLIP170,</t> or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).
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    (a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, CLIP170, or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).

    Journal: bioRxiv

    Article Title: A single-component optogenetic toolkit for programmable control of microtubule

    doi: 10.1101/2025.10.31.685931

    Figure Lengend Snippet: (a) Schematic of OptoMT design and its light-induced association with MT. (b) Domain architecture of OptoMT variants. The photosensitive module CRY2 (aa 1-498) was fused to tubulin-binding domains derived from Kinesin, EB1, CLIP170, or CAMSAP1. The optimal construct (variant 3, highlighted in red) exhibited minimal basal activity in the dark but strong MT binding upon blue light stimulation. (c) Confocal images of HeLa cells expressing the indicated mCh-OptoMT variants with or without blue light exposure (indicated by blue bars). Right, quantification of normalized MT-to-cytosol fluorescence intensity ratio before and after 1 s light stimulation (470 nm, 40 μW/mm²). n = 24 cells from three independent biological replicates. Also see Supplementary Videos 1-2 . (d) Confocal images showing precise spatiotemporal control of OptoMT labeling of MTs within the regions indicated upon blue light exposure (470 nm, 40 μW/mm²). Also see Supplementary Video 3 . (e) Confocal images showing robust and reversible OptoMT labeling of MTs across two successive dark-light cycles. Also see Supplementary Figure 1 . (f) Confocal images of HeLa cells expressing mCh-OptoMT (red) co-stained with anti-α-tubulin (green) and DAPI (blue). Cells were either kept in the dark (top) or illuminated with blue light (470 nm, 40 μW/mm 2 , 30 sec) before fixation and immunostaining. (g) Live-cell imaging of HeLa cells co-expressing GFP-OptoMT (green in the merged panel) and H2B-mCh (red), showing MT cytoskeleton and mitotic progression at different cell-cycle stages before and after blue light exposure (470 nm, 40 μW/mm 2 , 5 sec). (h) Confocal images of L3 stage C. elegans expressing GFP::α-tubulin and mCh::OptoMT in epithelia. Blue light induces MT binding by mCh::OptoMT. ( i - j ) Confocal images (i) and quantitative analysis (j) of OptoMT-mediated reversible MT labeling in C. elegans . See Supplementary Video 4 . The acquired data points were fitted by a single exponential decay function (t 1/2, on = 12.4 ± 3.2 sec; t 1/2, off = 252 ± 31 sec).

    Article Snippet: The plasmid templates for EB1 (#17234), CLIP170 (#54044), CAMSAP1 (# 59036),CAMSAP2 (#59037), KIF5A (#166954), and spastin (#134461) were purchased from Addgene.

    Techniques: Binding Assay, Derivative Assay, Construct, Variant Assay, Activity Assay, Expressing, Fluorescence, Control, Labeling, Staining, Immunostaining, Live Cell Imaging