Journal: PLoS ONE

Article Title: Up-Regulation of hERG K + Channels by B-RAF

doi: 10.1371/journal.pone.0087457

Figure Lengend Snippet: A. Representative original western blot showing hERG membrane protein abundance (anti-K v 11.1 antibody, Alamone Labs) analyzed by cell surface biotinylation in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (Control) or with 10 µM B-RAF inhibitor PLX-4720 (PLX-4720). B. Arithmetic means ± SEM (n = 7, arbitrary units) of normalized hERG membrane protein abundance analyzed by cell surface biotinylation in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 µM B-RAF inhibitor PLX-4720 (black bar). *(p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone. C. Representative original dot plots of hERG-FITC positive cells at the cell surface analysed by flow cytometry in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (Control) or with 10 µM B-RAF inhibitor PLX-4720 (PLX-4720); FL-1 Height: hERG-FITC fluorescence intensity. D. Arithmetic means ± SEM (n = 5, %) of normalized percentage of positive cells showing hERG expression at the cell surface analyzed by flow cytometry in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 µM B-RAF inhibitor PLX-4720 (black bar). *(p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone.

Article Snippet: After blocking with 5% non-fat dry milk in TBS 0.1% Tween20 for 1 hour at RT, the blots were incubated overnight at 4°C with rabbit anti-K v 11.1 (hERG, extracellular) antibody (diluted 1:200, Alamone Labs, Jerusalem, Israel).

Techniques: Western Blot, Flow Cytometry, Fluorescence, Expressing

Journal: Oncology Letters

Article Title: The combined use of EphA2/MMP-2 expression and MRI findings contributes to the determination of cerebral glioma grade

doi: 10.3892/ol.2019.10912

Figure Lengend Snippet: Expression of MMP-2 and EphA2 in cerebral glioma samples, according to the pathological grade of the tumor.

Article Snippet: One slide was routinely stained with hematoxylin and eosin in 25°C for 1–5 min. After blocking in BSA solution 37°C for 30 min (cat. no. P0260; Beyotime Institute of Biotechnology), the other slide was used for IHC staining using mouse anti-human MMP-2 and mouse anti-human EphA2 (Boster cat. no. M00286 and BM0833, both 1:200) monoclonal antibodies detected by streptavidin-peroxidase or 3′-diaminobenzidine visualization kits (OriGene Technologies, Inc.).

Techniques: Expressing

Journal: Oncology Letters

Article Title: The combined use of EphA2/MMP-2 expression and MRI findings contributes to the determination of cerebral glioma grade

doi: 10.3892/ol.2019.10912

Figure Lengend Snippet: Immunohistochemical staining of glioma tissues. (A) MMP-2 and (B) EphA2 expression was identified by immunofluorescence staining in the cytoplasm of glioma cells. Positive cells were stained brown. Percentages of stained cells are presented in the bar graphs. Data represent the mean ± standard deviation (n=5). ***P<0.001. Scale bar, 30 µm. EphA2, ephrin type-A receptor 2; MMP-2, metalloproteinase 2.

Article Snippet: One slide was routinely stained with hematoxylin and eosin in 25°C for 1–5 min. After blocking in BSA solution 37°C for 30 min (cat. no. P0260; Beyotime Institute of Biotechnology), the other slide was used for IHC staining using mouse anti-human MMP-2 and mouse anti-human EphA2 (Boster cat. no. M00286 and BM0833, both 1:200) monoclonal antibodies detected by streptavidin-peroxidase or 3′-diaminobenzidine visualization kits (OriGene Technologies, Inc.).

Techniques: Immunohistochemical staining, Staining, Expressing, Immunofluorescence, Standard Deviation

Journal: Oncology Letters

Article Title: The combined use of EphA2/MMP-2 expression and MRI findings contributes to the determination of cerebral glioma grade

doi: 10.3892/ol.2019.10912

Figure Lengend Snippet: Pearson's correlation analysis of the magnetic resonance imaging parameters and immunohistochemistry results. Scatterplots presenting the correlation between (A) MMP-2 and (B) EphA2 positivity with the EI; (C) MMP-2 and (D) EphA2 positivity with the EP; and (E) MMP-2 and (F) EphA2 positivity with maximum tumor diameter. EI, edema index; EP, enhancement percentage; EphA2, ephrin type-A receptor 2; MMP-2, metalloproteinase 2.

Article Snippet: One slide was routinely stained with hematoxylin and eosin in 25°C for 1–5 min. After blocking in BSA solution 37°C for 30 min (cat. no. P0260; Beyotime Institute of Biotechnology), the other slide was used for IHC staining using mouse anti-human MMP-2 and mouse anti-human EphA2 (Boster cat. no. M00286 and BM0833, both 1:200) monoclonal antibodies detected by streptavidin-peroxidase or 3′-diaminobenzidine visualization kits (OriGene Technologies, Inc.).

Techniques: Magnetic Resonance Imaging, Immunohistochemistry

Journal: Journal of Tissue Engineering

Article Title: Application of stem-cell media to explant culture of human periosteum: An optimal approach for preparing osteogenic cell material

doi: 10.1177/2041731413509646

Figure Lengend Snippet: Gene expression analysis of osteoblastic markers and bone-related growth factors of periosteal sheets expanded with 1% HS-STK1, 1% HS-STK1+3, and 10% FBS-M199 by qRT-PCR. (a) Runx2, (b) ALP, (c) col1a1, (d) OPN, (e) BMP-2, (f) RANKL, (g) VEGF-A, and (h) FGFR1. HS: human serum supplemented; FBS: fetal bovine serum; Runx2: runt-related transcription factor 2; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction; ALP: alkaline phosphatase; OPN: osteopontin; RANKL: receptor activator of nuclear factor kappa-B ligand; col1a1: collagen, type I, alpha 1; BMP: bone morphogenetic protein; VEGF-A: vascular endothelial growth factor-A; FGFR1: fibroblast growth factor receptor 1. n = 4, *p < 0.05, **p < 0.01, compared with controls.

Article Snippet: Sections were probed overnight at 4°C with the following primary antibodies diluted with blocking buffer: goat polyclonal anti-receptor activator of nuclear factor kappa-B ligand (RANKL; 1:100; Santa Cruz Biotechnology, Inc.), goat polyclonal anti-bone morphogenetic protein (BMP)-2/4 (1:100; R&D Systems Inc., Minneapolis, MN, USA), rabbit polyclonal anti-runt-related transcription factor 2 (RUNX2; 1:200; Abcam), or mouse monoclonal anti-vascular endothelial growth factor (VEGF) antibody (Abcam).

Techniques: Expressing, Quantitative RT-PCR, Polymerase Chain Reaction

Journal: Journal of Tissue Engineering

Article Title: Application of stem-cell media to explant culture of human periosteum: An optimal approach for preparing osteogenic cell material

doi: 10.1177/2041731413509646

Figure Lengend Snippet: (a–f) ALP activity and (g–i) immunoreactivity against OPN. (b and e) ALP activity was observed in the central part of CPSs expanded with 1% HS-STK1+3, (a and d) while the peripheral region of the 1% HS-STK1 sheets did not exhibit positive enzymatic activity. (c and f) The primary periosteal fragment region of the 10% FBS-M199 showed focal ALP activity. (h and k) OPN was consistently observed in the central part of the CPSs expanded with 1% HS-STK1+3, (g and j) while CPSs of 1% HS-STK1 did not exhibit any positive staining. (i) Focal positive staining of OPN was found in primary periosteal fragments of 10% FBS-M199. (m–x) IF of the other osteoblastic marker and growth factors (i.e. Runx2, BMP-2/4, RANKL, and VEGF) showed the strongest expression in CPSs of STK1+3, which is consistent with the results of mRNA expression by quantitative real-time PCR (upper half: IF; lower half: IF plus nuclear staining with DAPI). (a–c). Primary: region of the primary periosteal fragment and peripheral: area of the peripheral outgrowth of periosteal cells. Bar, 10 mm; (d–f and j–l) bar, 100 µm; (g–i) bar, 200 µm; and (m–x) bar, 40 µm. IF: immunofluorescence; ALP: alkaline phosphatase; CPS: cultured periosteal sheet; HS: human serum supplemented; Runx2: runt-related transcription factor 2; FBS: fetal bovine serum; OPN: osteopontin; RANKL: receptor activator of nuclear factor kappa-B ligand; BMP: bone morphogenetic protein; VEGF: vascular endothelial growth factor; mRNA: messenger RNA; PCR: polymerase chain reaction; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: Sections were probed overnight at 4°C with the following primary antibodies diluted with blocking buffer: goat polyclonal anti-receptor activator of nuclear factor kappa-B ligand (RANKL; 1:100; Santa Cruz Biotechnology, Inc.), goat polyclonal anti-bone morphogenetic protein (BMP)-2/4 (1:100; R&D Systems Inc., Minneapolis, MN, USA), rabbit polyclonal anti-runt-related transcription factor 2 (RUNX2; 1:200; Abcam), or mouse monoclonal anti-vascular endothelial growth factor (VEGF) antibody (Abcam).

Techniques: Activity Assay, Staining, Marker, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Cell Culture, Polymerase Chain Reaction

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: Characterization of 3CL pro cleavage specificity (A and B) MALDI-TOF-MS spectra of synthetic peptides spanning P4–P4’ of protein cleavage sites after incubation with 3CL pro (1:20 molar ratio, E:S). Product generation (red) and substrate consumption (black) were calculated as the peak area normalized to the total peak area in the spectrum. Apparent (app) k cat / K M values for 1 μM 3CL pro to convert 50% of substrate in 5, 15, 30, 60, 120 or 240 min are listed alongside bins of 4 peptides that share similar kinetic values arranged on a row-by-row basis. P4–P4’ sequence alignment using the Shapley color scale. Green protein names had cut sites identified by Edman sequencing of recombinant substrate digests. Boxed peptides, no cleavage. (C–I) Structures of the highest-ranked of 50,000 models of the active site of 3CL pro protomer 1 (PDB: 6XHM ) docked with P4–P4’ peptides from six 3CL pro substrates exhibiting a range of app k cat / K M values (circled in B). (C) 3CL pro dimer. Protomer 1, gray surface or green ribbons with catalytic Cys 145 shown. Protomer 2, orange surface with Ser 1 shown. Docking models with P4–P4’ peptide of: (C and D) RPAP1 (I_sc = −31.7), (E) IMA4 (I_sc = −30.6), (F) PTBP1 (I_sc = −31.7), (G) RBM15 (I_sc = −39.65), (H) MAP4K5 (I_sc = −28.1), and (I) CREB1 (I_sc = −30.3). Blue and red sticks, P and P’ amino acid residues, respectively. Yellow dashed sticks, hydrogen-bonds. See also Figure S2 .

Article Snippet: rabbit anti-SARS-CoV-1 3CL pro (1:2000) , Rockland , Cat# 200-401-A51; RRID: AB_828457.

Techniques: Incubation, Sequencing, Recombinant

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: 3CL pro cleavage sites and substrate proteins stringently identified in human embryonic kidney (HEK293) and human lung epithelial (BEAS-2B) cells, related to Figure 1

Article Snippet: rabbit anti-SARS-CoV-1 3CL pro (1:2000) , Rockland , Cat# 200-401-A51; RRID: AB_828457.

Techniques:

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: 3CL pro cleavage sites in human proteins identified by TAILS (A) Experimental design. Neo-N-termini of 3CL pro -cleaved substrates in HEK293 and BEAS-2B human lung epithelial cell lysates were isolated and identified by TAILS LC-MS/MS. Only those neo-N-termini in ≥ 2/3 HEK293 or ≥ 7/9 BEAS-2B independent cell experiments were considered for further substrate winnowing. (B) Classification of 1,649 quantified N-termini in N = 12 independent experiments. (C) 3CL pro candidate substrate cleavage site specificities (n = 292) versus other quantified neo-N-termini (n = 663). (D) Cellular distribution of high confidence cleavage sites (n = 102) in 101 human substrates after substrate winnowing . (E) Substrate Reactome gene set enrichment by hypergeometric distribution followed by FDR correction. Node radius designates gene enrichment; line widths are proportional to the overlap of shared substrates between connected nodes sharing ≥ 20% genes. See also Figure S1 and , , , Table S7. Comparison of interferon-treated BEAS-2B versus control. MSstats multiple sample t test results, related to Figure 1 , Table S8. Reactome gene sets enrichment in SARS-CoV-2 3CLpro substrates, related to Figure 1E .

Article Snippet: rabbit anti-SARS-CoV-1 3CL pro (1:2000) , Rockland , Cat# 200-401-A51; RRID: AB_828457.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Comparison, Control

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: 3CL pro cleaves RPAP1 and PTBP1, altering PTBP1 localization (A) Locations of 3CL pro cleavage sites in RPAP1 and PTBP1 identified by TAILS neo-N-terminal peptides (red) and Edman sequencing (green). Representative MS/MS spectra of cleaved neo-N-terminal peptides. (B) SDS-PAGE and Edman sequencing of human recombinant RPAP1 and PTBP1 incubated with 3CL pro +/− inhibitor GC376, or 3CL pro -C145A (1:5 mol/mol, E:S). Δ-substrate, no sequence obtained. (C) MALDI-TOF-MS kinetic analyses of 3CL pro cleavage of synthetic P4–P4’ peptides. (D) RPAP1 and PTBP1 immunoblots of primary HAECs lysates from 5 donors incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C. (E and F) (E) Immunoblots of 3CL pro , N-protein and (F) PTBP1 of Vero E6 cells infected with SARS-CoV-2 at a MOI 0.1 for 24 (n = 4) and 48 (n = 4) hpi, or mock (n = 3) ∗ , unspecific bands. (G) Subcellular localization of PTBP1 by confocal imaging. SARS-CoV-2-infected Vero E6 cells (N = 5, scale bar 50 μm). Cyan boxes, Spike-negative (S–) uninfected cells. Green boxes, Spike-positive (S+) infected cells. Enlarged detail of mock-infected, S+ and S– cells in the same field, scale bar 10 μm. (H) Nuclear to cytoplasmic ratio of PTBP1 was quantified for mock and Spike-positive SARS-CoV-2 infected cells. Statistical significance was assessed by Student’s t test (mean ± SD, N = 5, n = 51 cells, ∗ p ≤ 0.05). β-actin and β-tubulin loading controls. See also .

Article Snippet: rabbit anti-SARS-CoV-1 3CL pro (1:2000) , Rockland , Cat# 200-401-A51; RRID: AB_828457.

Techniques: Sequencing, Tandem Mass Spectroscopy, SDS Page, Recombinant, Incubation, Western Blot, Infection, Imaging

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: Hippo pathway substrate validation (A) 3CL pro cleavage sites in MAP4K5 and CREB1 identified by TAILS neo-N-terminal peptides (red) and Edman sequencing (green). Representative MS/MS spectra of cleaved neo-N-terminal peptides. (B) MALDI-TOF-MS kinetic analyses of 3CL pro cleavage of P4–P4’ peptides of MAP4K5 and CREB1. (C) SDS-PAGE, Edman sequencing (green) and immunoblot validation of human MAP4K5 and CREB1 substrates incubated with 3CL pro +/− inhibitor GC376, or 3CL pro -C145A (1:5 mol/mol, E:S). ΔMAP4K5 or ΔCREB1, no sequence obtained. (D and H) (D) YAP1, MAP4K5, CREB1 and (H) FYCO1 and FAF1 immunoblots of lysates from primary HAECs incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C. (E and F) (E) YAP1 and (F) MAP4K5 immunoblots of Vero E6 cells at 24 (n = 4) and 48 (n = 4) hpi (MOI 0.1) or mock (n = 3). MAP4K5 activity assay measured as ATP consumption using myelin basic protein as substrate. The area under the curve was calculated and compared by Student’s t test, (mean ± SD, n = 2, ∗∗ p ≤ 0.01). (G, I, and J) (G) Lysates of human Calu-3 lung cells infected with SARS-CoV-2 (MOI 0.1 and 1.0, n = 4, mock n = 2) were immunoblotted for (G) CREB1 48 hpi, (I) FYCO1 24 hpi and (J) FAF1 48 hpi. Statistical analysis of the relative amount of full-length protein (E and F) or proteolytic bands (G, I, and J) identified by molecular weights relative to β-actin was assessed by one-way ANOVA and Dunnett’s multiple comparisons test. Box and whiskers (min to max) plots, ∗∗∗ p ≤ 0.001, ∗∗ p ≤ 0.01, ∗ p ≤ 0.05, ns p > 0.05. β-actin and β-tubulin loading controls. See also and .

Article Snippet: rabbit anti-SARS-CoV-1 3CL pro (1:2000) , Rockland , Cat# 200-401-A51; RRID: AB_828457.

Techniques: Biomarker Discovery, Sequencing, Tandem Mass Spectroscopy, SDS Page, Western Blot, Incubation, Activity Assay, Infection

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: 3CL pro disrupts galectin-8 binding to Spike in antiviral-autophagy (A) Immunoblot of human galectin-8 (Gal8) in BEAS-2B cells in response to IFN- α , IFN-β, or vehicle. One way ANOVA and Dunnett's posthoc test (mean ± SD, n = 3 each, *** p ≤ 0.001, * p ≤ 0.05). (B) MALDI-TOF-MS of intact versus 3CL pro -cleaved synthetic Gal8 P4–P4’ peptide. (C) SDS-PAGE and Edman sequencing of Gal8 incubated with 3CL pro +/− inhibitor GC376, or 3CL pro C145A (1:5 mol/mol, E:S). (D) Structural model of Gal8 docked onto 3CL pro . 3CL pro cleavage site identified by the neo-N-terminal peptide (red) in 9/9 independent TAILS analyses. (E) Gal8 immunoblots of lysates from primary HAECs incubated with 3CL pro or 3CL pro -C145A (1:200 w/w, E:S) for 18 h, 37°C (N = 5). (F) Gal8 immunoblot of infected Calu-3 cells at 24 (n = 4) and 48 (n = 4) hpi (MOI 1.0, mock n = 3). β-actin and β-tubulin loading controls. (G) ELISA of SARS-CoV-2 Spike S1 protein binding intact Gal8 or 3CL pro -cleaved (ΔGal8) (mean ± SD, n = 2, N = 2, ∗∗∗∗ p ≤ 0.0001, ∗∗ p ≤ 0.01, ns p > 0.05, two-way ANOVA with Šídák’s multiple comparison test). (H) Immunoprecipitation (IP) by α-FLAG agarose-beads of HeLa cell lysates co-transfected with GFP-NDP52, WT-Gal8-FLAG or C-Gal8 (159-317)-FLAG. (I) Intact or cleaved Gal8 binding immobilized NDP52 detected with anti-C-Gal8 antibody. Student's t test, (mean ± SD, n = 3, N = 2, ns p > 0.05). (J) NDP52 binding Spike S1-associated intact or cleaved ΔGal8 (mean ± SD, n = 2, N = 2, ∗∗∗∗ p ≤ 0.0001, ∗∗∗ p ≤ 0.001, ∗ p ≤ 0.05, ns p > 0.05, two-way ANOVA with Šídák’s multiple comparison test). (K) Confocal microscopy of NDP52 and FLAG immunofluorescence after osmotic shock of HEK293 cells transfected with FLAG-tagged (WT) Gal8, N-Gal8 (1-158), or C-Gal8 (159-317). Scale bar, 20 μm. Quantification of NDP52 and Gal8-positive puncta, mean ± SD, n = 30, N = 5, ∗∗∗∗ p ≤ 0.0001, ns p > 0.05, one-way ANOVA with Tukey’s multiple comparison test. (L) Human lung tissue sections from normal subjects (N = 3) and post-mortem COVID-19 patients (N = 4) stained with hematoxylin and eosin (H&E), DAPI (blue) and immunofluorescence on the same sections for Gal8 (green), NDP52 (red), Spike (magenta) and merged image (orange). Scale bar, 50 μm. (M) Numbers of immunopositive cells for Gal8, NDP52 and Gal8 colocalized with NDP52 in healthy versus COVID-19 lung tissue. Total cell count distribution shown as a scatter dot plot, bar = median. See also Figure S6 .

Article Snippet: rabbit anti-SARS-CoV-1 3CL pro (1:2000) , Rockland , Cat# 200-401-A51; RRID: AB_828457.

Techniques: Binding Assay, Western Blot, SDS Page, Sequencing, Incubation, Infection, Enzyme-linked Immunosorbent Assay, Protein Binding, Comparison, Immunoprecipitation, Transfection, Confocal Microscopy, Immunofluorescence, Staining, Cell Counting

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet: Protein-protein interaction landscape of 3CL pro cell substrates (A) Network of direct (solid line) and physically associated (dashed line) interactors (blue dots) of the 101 3CL pro substrates (red circles) generated by the Intact-database (accessed May 2021). Interactors of ≥ 2 substrates are in magenta, only interactors of ≥ 3 substrates are labeled, the number of substrate interactions is indicated by circle size. Orange circles, candidate substrates manually annotated. (B) Top 5 CORUM protein complexes statistically enriched in the 3CL pro substrate-human interactome. (C) One-step direct neighborhood protein-protein interaction network of 3CL pro human substrates (n = 74, red circles) that are directly connected to SARS-CoV-2 proteins (n = 26, yellow diamonds) or connected via direct neighbors (n = 197). Assembled from IMex/Intact Coronavirus dataset accessed June 2, 2021. Yellow diamonds, viral proteins. Blue circles, direct interactors connecting viral proteins to substrates. Magenta circles, direct interactors connecting viral proteins to ≥ 2 substrates. Black edge, SARS-CoV-2/human protein interactions. See also .

Article Snippet: rabbit anti-SARS-CoV-1 3CL pro (1:2000) , Rockland , Cat# 200-401-A51; RRID: AB_828457.

Techniques: Generated, Labeling

Journal: Cell Reports

Article Title: Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CL pro substrate degradome

doi: 10.1016/j.celrep.2021.109892

Figure Lengend Snippet:

Article Snippet: rabbit anti-SARS-CoV-1 3CL pro (1:2000) , Rockland , Cat# 200-401-A51; RRID: AB_828457.

Techniques: Virus, Recombinant, Sequencing, Fluorescence, Modification, Protease Inhibitor, Polymer, Staining, Blocking Assay, Binding Assay, Activity Assay, Western Blot, Synthesized, Software, Control, Targeted Proteomics, Microscopy, Spectrophotometry, Mass Spectrometry