b0303 ic87114 pi3k c1 δ inhibitor  (Echelon Biosciences)

Journal: Immunity

Article Title: Six distinct NFκB signaling codons convey discrete information to distinguish stimuli and enable appropriate macrophage responses

doi: 10.1016/j.immuni.2021.04.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: TotalSeq ™ -B 0305 anti-mouse Hashtag Antibody , BioLegend , Cat# 155839; RRID:AB_2814071.

Techniques: Recombinant, Isolation, Enzyme-linked Immunosorbent Assay, Software, Cell Tracking Assay, Flow Cytometry

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: PI3K inhibition is required and sufficient for intracellular δR retention. (A) Representative confocal image from a fixed primary cultured TG neuron expressing a FLAG-tagged δR. Internal δR (red in merge) colocalizes with the Golgi marker GPP130 (green in merge). (B) Example image (of three independent experiments) of a PC12 cell showing surface δR. NGF treatment (60 min at 100 ng/μl) causes internal δR accumulation (arrow), colocalizing with TGN-38 (Golgi, green). (C) NGF-treated PC12 cells, chased with cycloheximide (CHX) for 1 h to prevent additional δR delivery, in the presence of inhibitors of MEK (10 µM U0126, denoted as U01), ROCK (5 µM Y-27632, denoted as Y-2), and PLC (10 µM U73122, denoted as U73). Percentage of cells with Golgi-localized δR >100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. ctrl). None of the inhibitors prevented NGF-mediated δR retention. (D) Representative images (of three independent experiments) showing that inhibition of PI3K with 10 μM Wtm or LY is sufficient to cause δR retention. (E) Quantitation of percentage of cells with δR Golgi localization showing that PI3K inhibition with Wtm or LY is sufficient for δR Golgi localization (>100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. NT). (F) Quantitation of the percentage of δR localization within the Golgi normalized to the total cell δR fluorescence, showing that PI3K inhibition with Wtm or LY is sufficient for δR retention (>100 cells each; mean ± SEM; *** p < 0.001). (G) Example images (of three independent experiments) for PC12 cells expressing FLAG-δR, untreated (left two columns) or treated with NGF (100 ng/ml) for 1 h. NGF treatment induces intracellular retention of δR (red), which colocalizes with the Golgi (green). Activation of PI3K by the p85 subunit–binding peptide 740Y PDGFR (50 µg/ml) decreased NGF-induced Golgi localization of δR. Images without and with 740Y PDGFR . (H) Quantitation of percentage of cells with δR Golgi localization, showing significant reduction in percentage of cells with Golgi-localized δR in the NGF condition after addition of the PI3K-activating peptide 740Y PDGFR (>100 cells each; mean ± SEM; ** p < 0.01 by one-way ANOVA with Dunn’s multiple comparison test). (I) Image analysis and quantification shows a significant reduction in percentage of total δR fluorescence that overlaps with the Golgi in the NGF condition after addition of PI3K-activating peptide 740Y PDGFR . 740Y PDGFR had no effect on Golgi localization of δR on its own (>100 cells each; mean ± SEM; *** p < 0.001 by one-way ANOVA with Dunn’s multiple comparison test).

Article Snippet: IC87114 , PI3K C1 δ inhibitor , 5 μM , Echelon Biosciences , B0305.

Techniques: Inhibition, Cell Culture, Expressing, Marker, Quantitation Assay, Fluorescence, Activation Assay, Binding Assay, Comparison

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: PI3K inhibition does not induce intracellular retention of μ R. (A) Representative images (of three independent experiments) of PC12 cells expressing FLAG- μ R as a control do not show retention after PI3K inhibition. (B) Quantitation of percentage of cells showing μ R retention after PI3K inhibition (>100 cells each; mean ± SEM; not significant [ p > 0.05] by two-sided t test vs. control). (C) Quantitation of percentage of total μ R fluorescence that overlaps with the Golgi after PI3K inhibition (>100 cells each; mean ± SEM; not significant [ p > 0.05] by two-sided t test vs. control). (D) Representative images (of three independent experiments) for δR endocytosis estimated by selectively labeling the surface vs. total pool of δR as described in Materials and Methods . PI3K inhibition did not cause endocytosis. The δR agonist DADLE was used as a control for endocytosis. (E) Pearson’s r of colocalization of the primary and secondary antibodies. High correlation denotes minimal endocytosis. DADLE significantly reduced the correlation, consistent with endocytosis (three representative fields; mean ± SEM; **** p < 0.0001 by two-sided t test vs. control).

Article Snippet: IC87114 , PI3K C1 δ inhibitor , 5 μM , Echelon Biosciences , B0305.

Techniques: Inhibition, Expressing, Control, Quantitation Assay, Fluorescence, Labeling

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: PI3K inhibition delays export of δR from the Golgi in HEK 293 cells. (A) Example images of HEK 293 cells expressing FLAG-δR (δR). In control conditions (ctrl), δR is present on the cell surface; however, in HEK 293 cells, PI3K inhibition by Wtm (10 µM) increases the Golgi localization of δR. (B) Image analysis and quantification show a significant increase in percentage of cells with the Golgi-localized δR after PI3K inhibition by 10 µM Wtm (>100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. ctrl) (C) Image analysis and quantification reveal a significant increase in percentage of total δR fluorescence that overlaps with the Golgi after PI3K inhibition by 10 µM Wtm (>100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. ctrl). (D) Example images of HEK 293 cells expressing FLAG- μ R. In control conditions (ctrl), μ R is present on the cell surface; however, unlike for δR, PI3K inhibition by 10 µM Wtm or LY does not increase the Golgi localization of μ R. (E) Image analysis and quantification resulted in a nonsignificant change in percentage of cells with the Golgi-localized μ R after PI3K inhibition by Wtm or LY (ctrl, n = 45; Wtm, n = 80; LY, n = 88; mean ± SEM; not significant by two-sided t test vs. ctrl). (F) Image analysis and quantification show a nonsignificant change in percentage of total μ R fluorescence that overlaps with the Golgi after PI3K inhibition by Wtm or LY (ctrl, n = 45; Wtm, n = 80; LY, n = 88; mean ± SEM; not significant by two-sided t test vs. ctrl). (G) Example time-lapse images (of four independent experiments) for HEK 293 cells expressing VSVG-δRtail-GFP imaged at the permissive temperature of 32°C. PI3K inhibition by 10 µM Wtm and LY resulted in sustained intracellular Golgi signal compared with control. (H) Quantification of the Golgi-associated fluorescence signal revealed a kinetic slowing of VSVG-δRtail-GFP trafficking from the Golgi after treatment with PI3K inhibitors Wtm and LY (control, 12 cells; Wtm, 14 cells; LY, 12 cells; mean ± SEM). (I) Quantification of the Golgi-associated VSVG fluorescence signal after 30 min, normalized to total, reveals that VSVG-δRtail-GFP requires the δRtail to show PI3K-dependent trafficking from the Golgi (control VSVG, 9 cells; VSVG LY294002, 9 cells; VSVG-δRtail control, 11 cells; VSVG-δRtail LY294002, 11 cells; mean ± SEM; *** p < 0.001).

Article Snippet: IC87114 , PI3K C1 δ inhibitor , 5 μM , Echelon Biosciences , B0305.

Techniques: Inhibition, Expressing, Control, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Class I PI3K is not required for Golgi retention of δR. (A) Example images (of three independent experiments) for PC12 cells expressing FLAG-δR, untreated (ctrl), or treated with NGF (100 ng/ml) for 1 h. NGF treatment induces intracellular retention of δR (red), which colocalizes with the Golgi (green). Inhibition of PI3K via 1 µM Wtm leads to partial accumulation of δR in the Golgi. Inhibition of PI3K via 10 µM Wtm leads to large accumulation of δR in the Golgi. (B) Image analysis and quantification show significant increase in percentage of cells with Golgi-localized δR after NGF treatment and PI3K inhibition by 1 and 10 µM Wtm (ctrl, 105 cells; NGF, 64 cells; 1 µM Wtm, 56 cells; 10 µM Wtm, 67 cells; mean ± SEM; ** p < 0.01, **** p < 0.0001 by two-sided t test vs. ctrl). (C) Further quantification demonstrated a significant increase in percentage of total δR fluorescence that overlaps with the Golgi in the NGF condition and a dose-dependent effect on percentage of δR-Golgi localization for 1 and 10 µM Wtm (ctrl, 105 cells; NGF, 64 cells; 1 µM Wtm, 56 cells; 10 µM Wtm, 67 cells; mean ± SEM; * p < 0.05, **** p < 0.0001 by two-sided t test vs. ctrl). (D) Example images (of three independent experiments) for PC12 cells expressing FLAG-δR, untreated (ctrl), or treated with NGF (100 ng/ml), LY (10 µM), or Wtm (10 µM) for 1 h induces intracellular retention of δR (red), which colocalizes with the Golgi (green). Class I–specific PI3K inhibitors PI-103 (PI3K C1 α, 50 nM), IC87114 (PI3K C1 δ, 5 µM), and AS605240 (PI3K C1 γ, 25 nM) were not sufficient to cause Golgi retention of δR. (E) Quantification demonstrated a significant increase in percentage of cells with Golgi-localized δR after NGF treatment and PI3K inhibition by LY (10 µM) or Wtm (10 µM) but not after class I–specific PI3K inhibition by PI3K inhibitors PI-103 (PI3K C1 α, 50 nM), IC87114 (PI3K C1 δ, 5 µM), and AS605240 (PI3K C1 γ, 25 nM; ctrl, 42 cells; NGF, 73 cells; LY, 77 cells; Wtm, 45 cells; α, 82 cells; δ, 99 cells; γ, 28 cells; mean ± SEM; *** p < 0.001 by one-way ANOVA with Dunn’s multiple comparison test). (F) Image analysis and quantification demonstrated a significant increase in percentage of cells with the Golgi-localized δR after NGF treatment and PI3K inhibition by 10 µM LY or Wtm but not after class I–specific PI3K inhibition by PI3K inhibitors PI-103 (PI3K C1 α, 50 nM), IC87114 (PI3K C1 δ, 5 µM), and AS605240 (PI3K C1 γ, 25 nM; ctrl, 42 cells; NGF, 73 cells; LY, 77 cells; Wtm, 45 cells; α, 82 cells; δ, 99 cells; γ, 28 cells; mean ± SEM; ** p < 0.01, **** p < 0.0001 by one-way ANOVA with Dunn’s multiple comparison test).

Article Snippet: IC87114 , PI3K C1 δ inhibitor , 5 μM , Echelon Biosciences , B0305.

Techniques: Expressing, Inhibition, Fluorescence, Comparison

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Class II PI3K C2A is required for surface trafficking of δR. (A) Immunoblotting for PI3K C2A (C2A) confirmed knockdown of C2A in PC12 cells stably expressing C2A lentiviral shRNA. Three different shRNA sequences were used to determine the most efficient knockdown. Representative immunoblot, with actin as a loading control. Blot densitometry was performed to quantitate the percentage of knockdown for C2A shRNA sequences 1–3 compared with control (Ctl) cells. (B) Densitometry (of three independent experiments) revealed C2A shRNA sequences 2 and 3 as providing significant reduction in C2A protein expression, with shRNA 3 providing the best knockdown at 49% (three independent experiments; mean ± SEM; ** p < 0.01 by two-sided t test vs. Ctl). (C) Example images (of three independent experiments) for PC12 cells expressing FAP-δR (δR) with and without lentiviral PI3K C2A shRNA 3 (C2A shRNA) with a GFP reporter. Cells stably expressing the PI3K C2A shRNA were identified via GFP expression and had increased intracellular δR. (D) Image analysis and quantification revealed a significant increase in percentage of total δR fluorescence that overlaps with the Golgi in cells stably expressing the PI3K C2A shRNA compared with δR-only cells (δR, 102 cells; δR + C2A shRNA, 75 cells; mean ± SEM; **** p < 0.0001 by two-sided t test vs. δR). (E) Further quantification confirmed a significant increase in percentage of cells with Golgi-localized δR in cells stably expressing the PI3K C2A shRNA compared with δR-only cells (δR, 102 cells; δR + C2A shRNA, 75 cells; mean ± SEM; **** p < 0.0001 by two-sided t test vs. δR). (F) Example images from PC12 FAP- δR cells expressing EPAC cAMP biosensor and control nontargeted siRNA. Images reveal an increase in cAMP EPAC FRET ratio over time after 5 µM forskolin addition and subsequent decrease in cAMP and receptor internalization after δR agonist DADLE (10µM) addition. (G) Quantitative analysis of the EPAC FRET ratio, showing an increase in cAMP after forskolin addition and decrease after δR agonist DADLE addition in control (Ctl) siRNA cells. Similar experiments performed in cells transfected with PI3K C2A siRNA exhibited no decrease in cAMP after δR agonist DADLE (Ctl siRNA, 12 cells; C2A siRNA, 10 cells; mean ± SEM). (H) Analysis of percentage of cAMP inhibition after δR agonist addition demonstrates PI3K C2A siRNA ability to significantly reduce cAMP inhibition compared with control siRNA cells (Ctl siRNA, 12 cells; C2A siRNA, 10 cells; mean ± SEM; **** p < 0.0001 by two-sided t test Ctl vs. C2A siRNA).

Article Snippet: IC87114 , PI3K C1 δ inhibitor , 5 μM , Echelon Biosciences , B0305.

Techniques: Western Blot, Knockdown, Stable Transfection, Expressing, shRNA, Control, Fluorescence, Transfection, Inhibition

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Expression of class II PI3K C2A is sufficient to induce surface trafficking of δR. (A) Example images (of three independent experiments) for PC12 cells expressing GFP-PI3K C2A (green) demonstrate a pool of GFP-PI3K C2A enriched around the Golgi (TGN-38, red) in control conditions (Ctl) that is reduced after 1 h of treatment with NGF (100 ng/ml). (B) Quantification of GFP-PI3K C2A mean fluorescence intensity revealed a significant decrease in Golgi localization after NGF treatment (control, 36 cells; NGF, 45 cells; mean ± SEM; **** p < 0.0001 by two-sided t test vs. control). (C) Additional quantification of the observed shift in GFP-PI3K C2A distribution shows a shift from perinuclear localization to diffuse cytoplasmic localization after NGF treatment (control, 28 of 36 perinuclear; NGF, 12 of 33 perinuclear; mean ± SEM; **** p < 0.0001 by two-sided Fisher’s exact test vs. control). (D) Example images (of three independent experiments) for PC12 cells expressing FAP-δR (δR, red) alone and in combination with overexpression of GFP-PI3K C2A (PI3K C2A, green) untreated (Ctl) or treated with NGF (100 ng/ml) for 1 h. Overexpression of GFP-PI3K C2A was sufficient to prevent the NGF-induced Golgi retention of δR. (E) Quantification and image analysis show a significant decrease in percentage of total δR fluorescence that overlaps with the Golgi in cells treated with NGF when overexpressing GFP-PI3K C2A compared with δR-only cells (>100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. δR Ctl). (F) Further quantification demonstrated a significant decrease in percentage of cells with Golgi-localized δR in cells treated with NGF when overexpressing GFP-PI3K C2A compared with δR-only cells (>100 cells each; mean ± SEM; **** p < 0.0001 by two-sided t test vs. δR Ctl). (G) Example image showing GFP-PI3K C2A (green) expression in primary TG neuron counterstained live with Hoechst DNA stain (blue). TG neurons expressing GFP-PI3K C2A have ∼3.27-fold higher deltorphin II–Cy3 binding compared with nonexpressing (without PI3K-C2A) TG neurons (without PI3K-C2A, 33 cells; expresses PI3K-C2A, 35 cells; mean ± SEM; **** p < 0.0001 by two-sided t test vs. without PI3K-C2A).

Article Snippet: IC87114 , PI3K C1 δ inhibitor , 5 μM , Echelon Biosciences , B0305.

Techniques: Expressing, Control, Fluorescence, Over Expression, Staining, Binding Assay

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Optogenetic recruitment of the PI3K C2A kinase domain to the TGN is sufficient to induce δR surface trafficking. (A) Schematic showing the optogenetic recruitment strategy for targeting the PI3K C2A kinase domain to the TGN upon stimulation with blue light. The CRY2-CIBN optical recruitment system was adapted by cloning the PI3K C2A kinase domain (amino acids 863–1405, KD) onto the C-terminus of an mCherry-CRY2 chimeric construct to make mCherry-CRY2-PI3K-C2A-KD. In addition, the GFP-CIBN was adapted for TGN-specific recruitment by cloning the N-term of 2,6-sialyltransferase onto the C-terminus of GFP-CIBN to make GFP-CIBN-SialyT. The GFP-CIBN-SialyT has a basal TGN localization and the mCherry-CRY2-PI3K-C2A-KD is mostly cytoplasmic before recruitment. Optorecruitment is activated by blue light stimulation at 488 nm driving the mCherry-CRY2-PI3K-C2A-KD to the TGN. (B) PC12 cells were transiently transfected with the mCherry-CRY2-PI3K-C2A-KD (C2A KD, red), GFP-CIBN-SialyT (CIBN-TGN, green), and the TGN-localized PI(4)P sensor PH-FAPP1-iRFP (PH-FAPP1, blue) for live imaging of the optogenetic recruitment. During the live stimulation with 488-nm light, recruitment of the C2A KD to the CIBN-TGN and PH-FAPP1 can be observed within 5 s. (C) Analysis of the recruitment and colocalization of the C2A KD to the CIBN-TGN and PH-FAPP1 region was performed via a line-scan plot (example line in yellow) to identify relative spatiotemporal fluorescence intensity over a pixel area. Over time, the peak fluorescence intensity and the fluorescence intensity distribution for C2A KD (red), CIBN-TGN (green), and PH-FAPP1 (blue) are all highly overlapping after 5 s. (D) PC12 cells were transiently transfected with the mCherry-CRY2-PI3K-C2A-KD (C2A KD, blue), GFP-CIBN-SialyT (CIBN-TGN, green), and FAP-δR (δR, red) for live imaging of the optogenetic recruitment and induced surface trafficking after NGF-induced retention of δR. NGF (100 ng/ml) was added for 1 h to cause Golgi retention of δR (yellow arrow). Optical recruitment of the mCherry-CRY2-PI3K-C2A-KD to the TGN was performed to stimulate PI3K C2A and induce surface trafficking of the retained pool of δR. After recruitment, the Golgi pool of δR decreases and appears to vesiculate. (E) Analysis of live-imaging frames reveals the formation of vesicles containing δR beginning in the TGN (yellow arrow), bud off, and appear to be trafficking to the cell surface. (F) Image analysis and quantification of the complete recruitment movie demonstrate a decrease in the δR fluorescence intensity within the TGN over time after active and sustained recruitment of the mCherry-CRY2-PI3K-C2A-KD to the TGN. (G) Quantification and analysis (of eight independent experiments) show the fold change in δR membrane and TGN-localized fluorescence normalized to their starting values (dotted blue line) after recruitment of mCherry-CRY2-PI3K-C2A-KD to the TGN. There is a significant decrease in the fluorescence intensity of δR within the TGN after recruitment (TGN δR, 37%), as well as a significant increase in total δR membrane fluorescence (membrane δR, 7%; membrane δR, eight cells; TGN δR, eight cells; mean ± SEM; **** p < 0.0001 by two-sided t test vs. respective Ctl).

Article Snippet: IC87114 , PI3K C1 δ inhibitor , 5 μM , Echelon Biosciences , B0305.

Techniques: Cloning, Construct, Transfection, Imaging, Fluorescence, Membrane

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Pharmacological compounds used throughout the article.

Article Snippet: IC87114 , PI3K C1 δ inhibitor , 5 μM , Echelon Biosciences , B0305.

Techniques: Concentration Assay

Journal: Molecular Biology of the Cell

Article Title: PI3K class II α regulates δ-opioid receptor export from the trans -Golgi network

doi: 10.1091/mbc.E17-01-0030

Figure Lengend Snippet: Pharmacological compounds used throughout the article.

Article Snippet: IC87114 , PI3K C1 δ inhibitor , 5 µM , Echelon Biosciences , B0305.

Techniques: Concentration Assay

Journal: Autophagy

Article Title: The human CD5L/AIM-CD36 axis: A novel autophagy inducer in macrophages that modulates inflammatory responses

doi: 10.1080/15548627.2015.1017183

Figure Lengend Snippet: CD5L increases class III PtdIns3K. ( A ) THP1 MФ (top) and PB monocytes (bottom) preincubated with 1 μg/ml r-HsCD5L or ALB for 24 h were treated for 45 min with PtdIns3K inhibitors at the following concentrations: 1 μM Pi-103 (Pi-103, PI3K/p110α, β, γ, δ inhibitor), 1 μM IC87114 (IC, PI3K/p110δ inhibitor), 0.1 mM 3-MA (3-MA, PI3K/PtdIns3K inhibitor), 200 μM LY294002 (Ly, pan-PI3K/PtdIns3K inhibitor), 10 μM wortmannin (W, pan-PI3K/PtdIns3K inhibitor), or with DMSO as control. Cells were then incubated for 4 h with 100 ng/ml Pam3CSK4 or 100 ng/ml LPS, and culture supernatant fractions were collected and assayed for TNF production by ELISA. Mean values ± SEM from 4 independent experiments (for THP1 MФ) or PB monocytes obtained from 3 healthy donors performed in triplicate are shown. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 Student t test. ( B ) To assay PtdIns3P cellular content, THP1 MФ (left) and PB monocytes (right) preincubated with 1 μg/ml r-HsCD5L or ALB for 24 h were treated for 45 min with 0.1 mM 3-MA (3-MA) or DMSO as control, and then incubated with 1 μg/ml Pam3CSK4 for 30 min when indicated. PtdIns3P was stained with a specific antibody (green) and nuclei with Hoechst dye (blue). Upper panel: representative confocal microscopy images of THP1 MФ (left) and PB monocytes (right) . Lower panel: mean PtdIns3P fluorescence intensity values ± SEM in more than 20 cells from independent experiments scored in random fields. * P ≤ 0.05; *** P ≤ 0.001, one-way ANOVA.

Article Snippet: In experiments performed in the presence of signaling inhibitors, these compounds were added 45 min prior to TLR stimulation at the following concentrations: 20 μM SB203580, 50 μM SP600125, 100 μM PD98059, 10 μM wortmannin, 200 μM LY294002 (InvivoGen, tlrl-sb20, tlrl-sp60, tlrl-pd98, tlrl-wtm and tlrl-ly29, respectively), 1 μM Pi-103, 1 μM IC87114 (from Echelon Bioscience Inc., b-0303/b-0305/b-0301), and 0.1 mM 3-methyladenine (Sigma-Aldrich, M9281).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Staining, Confocal Microscopy, Fluorescence