Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Assessment of Lung Eosinophils In Situ Using Immunohistological Staining

doi: 10.1007/978-1-0716-1001-5_17

Figure Lengend Snippet: Allergen-challenged FFPE lung sections with EPX fluorescent IHC with TSA. (a) Eosinophils are stained for EPX with Cy3-conjugated tyramide substrate (orange). Nuclei are counterstained with DAPI (blue). (b) Negative control without the primary antibody. Image was acquired with a Plan-Apochromat ×63 objective on a Zeiss LSM 800 microscope

Article Snippet: Eosinophils are stained for EPX (red) and nuclei are counterstained with DAPI (blue). ( a ) Tile (5 × 5) image was acquired with a Plan-Apochromat ×63 objective on a Zeiss LSM 800 microscope. ( b ) Zoomed in image of ( a ). ( c ) Negative control without the primary antibody

Techniques: Staining, Negative Control, Microscopy

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Assessment of Lung Eosinophils In Situ Using Immunohistological Staining

doi: 10.1007/978-1-0716-1001-5_17

Figure Lengend Snippet: Allergen-challenged FFPE lung sections with EPX indirect IF. Eosinophils are stained for EPX (red) and nuclei are counterstained with DAPI (blue). (a) Tile (5 × 5) image was acquired with a Plan-Apochromat ×63 objective on a Zeiss LSM 800 microscope. (b) Zoomed in image of (a). (c) Negative control without the primary antibody

Article Snippet: Eosinophils are stained for EPX (red) and nuclei are counterstained with DAPI (blue). ( a ) Tile (5 × 5) image was acquired with a Plan-Apochromat ×63 objective on a Zeiss LSM 800 microscope. ( b ) Zoomed in image of ( a ). ( c ) Negative control without the primary antibody

Techniques: Staining, Microscopy, Negative Control

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Assessment of Lung Eosinophils In Situ Using Immunohistological Staining

doi: 10.1007/978-1-0716-1001-5_17

Figure Lengend Snippet: MBP and EPX dual fluorescent ICC. Cells were prepared by cytocentrifugation and then stained for both EPX and MBP. Eosinophils are stained for EPX (red) and MBP (green). Nuclei are counterstained with DAPI (blue). Image was acquired with a Plan-Apochromat ×63 objective using Airyscan on a Zeiss LSM 800 microscope

Article Snippet: Eosinophils are stained for EPX (red) and nuclei are counterstained with DAPI (blue). ( a ) Tile (5 × 5) image was acquired with a Plan-Apochromat ×63 objective on a Zeiss LSM 800 microscope. ( b ) Zoomed in image of ( a ). ( c ) Negative control without the primary antibody

Techniques: Staining, Microscopy

Journal: Innate immunity

Article Title: Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

doi: 10.1177/1753425915586075

Figure Lengend Snippet: Figure 6. Lyn is essential to the activation of PI 3-kinase through phosphorylation of its p110 subunit. (A; Supplementary movies M1–M4) HEK 293–TLR2 cells were transfected with a fusion protein combining GFP and the PH domain of AKT. Transfected cells were either co-transfected with LynDK (500 ng/ml) or incubated with PP2. Cells were then stimulated with Pam3 (100 ng/ml) and the kinetics of the AKT–PH construct was observed by microvideoscopy using Zeiss Axiovert inverted microscope equipped with the Metafluor imaging system. Presented here are the images corresponding to 15 min of Pam3 stimulation. Controls correspond to cells incubated with DMSO and pcDNA empty vector, or cells incubated with LY294002 (25 mM), a specific inhibitor of PI 3-kinase. (B) HEK 293–TLR2 cells were transfected with pcDNA vector (vehicle) or LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml). Lysates were immunoprecipitated with anti-Flag Abs and recruitment of PI 3-kinase to TLR2 was observed by Western blot with anti-p85a Abs. (C) Tyrosine phosphorylation of the p85a subunit was evaluated by Western blot in HEK 293–TLR2 transfected with LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml). Anti-phosphotyrosine (4G10 clone) and anti-p85a Abs were used for immuno- precipitation and Western blot. (D) HEK 293–TLR2 were transfected with LynDK (500 ng/ml) and stimulated with Pam3 (100 ng/ml) and lysates were immunoprecipitated with either 4G10 or anti-p110 Abs. Phosphorylation of p110 was then revealed by Western blot with anti-p110 or 4G10 Abs. Controls correspond to cells transfected with pcDNA empty vector. (E) THP1–CD14 cells were incubated with PP2 (25mM) or DMSO, stimulated with Pam3 (100 ng/ml) and lysed. Phosphorylation of p110 catalytic subunit of PI 3-kinase was revealed by Western blot of THP1–CD14 lysates immunoprecipitated with either 4G10 or anti-p110 Abs. Controls correspond to cells treated with DMSO. These results are representative of three independent experiments.

Article Snippet: Polyclonal Abs to phospho-AKT (Ser473), AKT, phospho-P65 (Ser536), P65, phosphoP38, phospho-ERK, phospho-SAP-JNK, ERK, SAPJNK, IkB and mAb P38 were from Cell Signaling (Danvers, MA, USA). mAb to Flag was from Sigma. mAbs against CD14 and aminoacid 800-1139 of human p110 isoforms were from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Transfection, Incubation, Construct, Inverted Microscopy, Imaging, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Innate immunity

Article Title: Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

doi: 10.1177/1753425915586075

Figure Lengend Snippet: Figure 7. Lyn controls the PI 3-kinase pathway through phosphorylation of its p110 subunit after TLR2 engagement. The presence of bacteria-derived acylated lipoproteins results in the heterodimerization of TLR2 with TLR1/6 in membrane microdomains. MyD88/ IRAK are recruited to the receptor and lead to degradation of IkB and translocation of NF-kB subunits. A Rac/PI 3-kinase-dependent signaling pathway has also been described, including CD14 and Lyn that contribute to the activation cluster. After tyrosine phos- phorylation of the cytoplasmic domain of TLR2, the p85a subunit of PI 3-kinase is recruited to the receptor and phosphorylated on tyrosine. A Lyn-dependent tyrosine-phosphorylation is required to activate the PI 3-kinase catalytic subunit p110 that allows for recruitment of AKT to the inner membrane, precluding a cascade that results in transactivation of the p65 subunit of NF-kB. This leads to the nuclear translocation of a functional p50/p65 NF-kB heterodimer that results in the gene expression of pro-inflammatory cytokines.

Article Snippet: Polyclonal Abs to phospho-AKT (Ser473), AKT, phospho-P65 (Ser536), P65, phosphoP38, phospho-ERK, phospho-SAP-JNK, ERK, SAPJNK, IkB and mAb P38 were from Cell Signaling (Danvers, MA, USA). mAb to Flag was from Sigma. mAbs against CD14 and aminoacid 800-1139 of human p110 isoforms were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Bacteria, Derivative Assay, Membrane, Translocation Assay, Activation Assay, Functional Assay, Gene Expression

Journal: Nature Communications

Article Title: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth

doi: 10.1038/s41467-020-18753-6

Figure Lengend Snippet: a PINCH-1 KO A549 cells were analyzed by Western blotting with antibodies as indicated. The levels of DRP1, FIS1, MFF, short form of OPA1(S-OPA1), long form of OPA1(L-OPA1), MFN1 and MFN2 in PINCH-1 KO A549 cells were quantified by densitometry and compared to those in A549 cells (normalized to 1) (right, n = 3). b PINCH-1 KO A549 cells were infected with lentiviral vectors encoding 3f-P1 or 3 f. Three days later, cells (as indicated) were analyzed by Western blotting. DRP1 level in indicated samples was quantified by densitometry and compared to A549 (right, n = 3). c H1299 cells were infected with Sh-P1 or Sh-con lentivirus and analyzed by Western blotting with antibodies as indicated. The levels of DRP1 in PINCH-1 knockdown H1299 cells were quantified by densitometry and compared to those in H1299 cells (normalized to 1) (right, n = 5). d DRP1 mRNA levels in A549 cells (as indicated) were analyzed by RT-PCR ( n = 3). e DRP1 mRNA levels in H1299 cells (as indicated) were analyzed by RT-PCR ( n = 6). f A549 cells were infected with ILK shRNA (Sh-ILK) or Sh-con lentivirus and analyzed by Western blotting as indicated. The levels of DRP1 in ILK knockdown cells were quantified by densitometry and compared to those in A549 cells (normalized to 1) (right, n = 4). g Mitochondria were stained with MitoTracker Red CMXRos and the percentages of mitochondria with different morphologies were quantified (right, n = 30 cells). Bar, 5 μm. h Mitochondrial areas in z-stack images were quantified ( n = 31 cells). i Wild type and kindlin2 (K2) KO A549 cells were analyzed by Western blotting with antibodies recognizing DRP1, P1 or tubulin. The level of DRP1 in kindlin2 KO A549 cells was quantified by densitometry and compared to that in wild type A549 cells (normalized to 1) (right, n = 4). Data represent mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis. * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance. Source data are provided as a Source Data file. The samples in a , b , c , f , and i were from the same experiment and the blots were processed in parallel.

Article Snippet: Immunohistochemistry was performed using the MaxVision TM HRP-Polymer anti-Rabbit IHC Kit (MXB biotechnologies) with rabbit antibodies against PINCH-1 (Abcam, ab108609,1:800), PYCR1 (Proteintech,13108-1-AP,1:800), DRP1 (Proteintech,12957-1-AP,1:800), Ki67 (CST,12202P, 1:1000) or collagen 1A1 (Novus,NB600-408).

Techniques: Western Blot, Infection, Knockdown, Reverse Transcription Polymerase Chain Reaction, shRNA, Staining

Journal: Nature Communications

Article Title: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth

doi: 10.1038/s41467-020-18753-6

Figure Lengend Snippet: a PINCH-1 KO A549 cells were transfected with siRNA targeting DRP1 (Si-DRP1) or control siRNA (Si-con) as indicated. Three days later, the cells were analyzed by Western blotting with antibodies recognizing DRP1, P1, or tubulin. The samples were from the same experiment and the blots were processed in parallel. Mitochondria were stained with MitoTracker Red CMXRos ( b ) and the percentages of mitochondria with different morphologies were quantified ( c ) (A549 n = 43 cells, P1KO n = 70 cells, P1KO + Si-Con n = 77 cells, P1KO + Si-DRP1 n = 55cells; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-con P < 0.0001). Bar, 5μm. d Mitochondrial areas in z-stack images were quantified (A549 and P1KO and P1KO + Si-con n = 32 cells, P1KO + Si-DRP1 n = 30 cells; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-con P < 0.0001). e , f Mitochondria (red arrows) were observed under TEM ( e , bar, 500 nm) and the areas of mitochondria were quantified ( f , n = 40 mitochondria; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-con P = 0.0028). The cells were stained with DAPI (blue) and antibody for Ki67 (purple) ( g ) and the percentages of Ki67-positive cells were quantified ( h , A549 and P1KO n = 33 fields, P1KO + Si-con and P1KO + Si-DRP1 n = 31 fields; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-con P < 0.0001). Bar, 25 μm. i The cells were cultured for 4 days and the numbers of live cells were analyzed as in Fig. ( n = 4; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-con P < 0.0001). Data in c , d , f , h , and i represent mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis, ** P < 0.01; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed using the MaxVision TM HRP-Polymer anti-Rabbit IHC Kit (MXB biotechnologies) with rabbit antibodies against PINCH-1 (Abcam, ab108609,1:800), PYCR1 (Proteintech,13108-1-AP,1:800), DRP1 (Proteintech,12957-1-AP,1:800), Ki67 (CST,12202P, 1:1000) or collagen 1A1 (Novus,NB600-408).

Techniques: Transfection, Control, Western Blot, Staining, Cell Culture

Journal: Nature Communications

Article Title: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth

doi: 10.1038/s41467-020-18753-6

Figure Lengend Snippet: a PINCH-1 KO A549 cells were transfected with Si-DRP1 or Si-con as indicated. Three days later, cells (as indicated) were analyzed by Western blotting with antibodies recognizing PYCR1, DRP1, PINCH-1, or tubulin. PYCR1 level was quantified by densitometry (right, n = 4; A549 vs P1KO P = 0.0076, A549 vs P1KO + Si-con P = 0.0023, P1KO vs P1KO + Si-DRP1 P = 0.0419, P1KO + Si-con vs P1KO + Si-DRP1 P = 0.012). b The cytosolic (Cyto) (lane 5, 7, 9 and 11), mitochondrial (Mito) (lane 6, 8, 10, and 12) and total (lane 1, 2, 3, and 4) fractions from the cells were analyzed by Western blotting with antibodies recognizing kindlin-2 (K2), PHB2, or tubulin. Kindlin-2 level in mitochondria was quantified (right, n = 3; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-DRP1 P < 0.0001). c Cells were analyzed by PLA with kindlin-2 and PYCR1 antibodies. Bar, 10 μm. The number of PLA dots per cell were counted (right, n = 40 cells; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-DRP1 P < 0.0001). d Cells (as indicated) were analyzed by IP and Western blotting. Lane 2, the sample was prepared as that of lane 3 except anti-kindlin-2 antibody was substituted with irrelevant mouse IgG. PYCR1 level was quantified by densitometry (right, n = 4; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P < 0.0001, P1KO + Si-con vs P1KO + Si-DRP1 P < 0.0001). e The proline level was analyzed using the absorbance method as described in the “Methods” (right, n = 3 independent experiments; A549 vs P1KO P < 0.0001, A549 vs P1KO + Si-con P < 0.0001, P1KO vs P1KO + Si-DRP1 P = 0.0086, P1KO + Si-con vs P1KO + Si-DRP1 P = 0.0058). A representative set of samples were shown in the left. Bar, 1 cm. f A549 cells were infected with lentiviral vector encoding 3xFLAG-tagged MFN2 (3f-MFN2) or with 3xFLAG vector (3f). Three days later, the cells were analyzed by PLA with kindlin-2 and PYCR1 antibodies. Bar, 10 μm. The number of PLA dots per cell were counted (right, A549 n = 34 cells, 3f n = 33 cells, 3f-MFN2 n = 46 cells; A549 vs 3f-MFN2 P = 0.0074, 3f vs 3f-MFN2 P = 0.0027). g The cytosolic (lane 4, 6, and 8), mitochondrial (lane 5, 7, and 9) and total (lane 1, 2, and 3) fractions from the cells were analyzed by Western blotting. The levels of kindlin-2 in the cytosolic or mitochondrial fractions were quantified by densitometry. Right panel, the ratio of the mitochondrial kindlin-2 level divided by the cytosolic kindlin-2 level in the MFN2 overexpressing cells was compared to that in the control infectants or wild type A549 cells (normalized to 1, n = 3; A549 vs 3f-MFN2 P < 0.0001, 3f vs 3f-MFN2 P < 0.0001). Data in a – g represent mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis, * P < 0.05; ** P < 0.01; *** P < 0.001. The samples in a , b , d , and g were from the same experiment and the blots were processed in parallel. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed using the MaxVision TM HRP-Polymer anti-Rabbit IHC Kit (MXB biotechnologies) with rabbit antibodies against PINCH-1 (Abcam, ab108609,1:800), PYCR1 (Proteintech,13108-1-AP,1:800), DRP1 (Proteintech,12957-1-AP,1:800), Ki67 (CST,12202P, 1:1000) or collagen 1A1 (Novus,NB600-408).

Techniques: Transfection, Western Blot, Infection, Plasmid Preparation, Control

Journal: Nature Communications

Article Title: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth

doi: 10.1038/s41467-020-18753-6

Figure Lengend Snippet: a PINCH-1 KO A549 cells were infected with 3xFLAG-PYCR1 (3f-PY1) or 3xFLAG (3f) lentivirus. The cells were analyzed by Western blotting with antibodies as indicated. DRP1 level was quantified (right, n = 3; A549 vs P1KO P = 0.006, A549 vs P1KO + 3f P = 0.013, P1KO vs P1KO + 3f-PY1 P = 0.019, P1KO + 3f vs P1KO + 3f-P1 P = 0.0434). The samples were from same experiment and blots were processed in parallel. b The proline level was analyzed using the absorbance method (right, n = 3 independent experiments; A549 vs P1KO P < 0.0001, A549 vs P1KO + 3f P < 0.0001, P1KO vs P1KO + 3f-PY1 P < 0.0001, P1KO + 3f vs P1KO + 3f-P1 P < 0.0001). A representative set of samples were shown in the left. Bar, 1 cm. c The numbers of live cells (A549 cells, red line; P1 KO cells, orange line; 3f lentiviral vector infected P1 KO cells, blue line; 3f-PY1 lentiviral vector infected P1 KO cells, green line) were analyzed as in Fig. ( n = 3). d Cells were stained with DAPI (blue) and Ki67 antibody (purple). Bar, 25 μm. The percentages of Ki67 positive cells were quantified (right, A549 n = 56 fields, P1KO n = 33 fields, P1KO + 3f n = 42 fields, P1KO + 3f-PY1 n = 51 fields; A549 vs P1KO P < 0.0001, A549 vs P1KO + 3f P < 0.0001, P1KO vs P1KO + 3f-PY1 P < 0.0001, P1KO + 3f vs P1KO + 3f-P1 P < 0.0001). e Mitochondria were stained with MitoTracker Red CMXRos and mitochondria with different morphologies were quantified (right, A549 and P1KO n = 50 cells, P1KO + 3f n = 40 cells, P1KO + 3f-PY1 n = 37 cells; A549 vs P1KO P < 0.0001, A549 vs P1KO + 3f P < 0.0001, P1KO vs P1KO + 3f-PY1 P < 0.0001, P1KO + 3f vs P1KO + 3f-P1 P < 0.0001). Bar, 5 μm. f Mitochondrial areas in z-stack images were quantified (A549 n = 33 cells, P1KO n = 35 cells, P1KO + 3f n = 36 cells, P1KO + 3f-PY1 n = 37 cells; A549 vs P1KO P < 0.0001, A549 vs P1KO + 3f P < 0.0001, P1KO vs P1KO + 3f-PY1 P < 0.0001, P1KO + 3f vs P1KO + 3f-P1 P < 0.0001). g Mitochondria (red arrows) were observed under TEM (left panels, bar, 500 nm) and mitochondria areas were quantified (right panel, n = 40 mitochondria; A549 vs P1KO P < 0.0001, A549 vs P1KO + 3f P < 0.0001, P1KO vs P1KO + 3f-PY1 P < 0.0001, P1KO + 3f vs P1KO + 3f-P1 P = 0.001). Data in a – g represent mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey–Kramer post-hoc analysis, * P < 0.05; *** P < 0.001. Source data are provided in Source Data file.

Article Snippet: Immunohistochemistry was performed using the MaxVision TM HRP-Polymer anti-Rabbit IHC Kit (MXB biotechnologies) with rabbit antibodies against PINCH-1 (Abcam, ab108609,1:800), PYCR1 (Proteintech,13108-1-AP,1:800), DRP1 (Proteintech,12957-1-AP,1:800), Ki67 (CST,12202P, 1:1000) or collagen 1A1 (Novus,NB600-408).

Techniques: Infection, Western Blot, Plasmid Preparation, Staining

Journal: Nature Communications

Article Title: PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth

doi: 10.1038/s41467-020-18753-6

Figure Lengend Snippet: The lung of the mice was administrated with Ad-Cre and analyzed 16 weeks later. a Sections of the lung tissues from the mice (as specified in the figure) were analyzed by immunostaining with antibodies for PINCH-1 (P1)(top), DRP1 (middle), or PYCR1 (bottom). Bar, 20 μm. The boxed areas in the IHC staining were enlarged and shown in the upper right corner. Right panels, the mean intensities of PINCH-1, DRP1, and PYCR1 staining in the Kras LSL−G12D/+ ; PINCH-1(P1) fl/fl (Kras fl/+ ; P1 fl/fl ) group were quantified and compared to those of the Kras fl/+ group (normalized to 1; n ≥ 30 fields from 6 mice for each group; different mice in each group were coded with different colors; P < 0.0001). b The DRP1 mRNA levels from the lung tissues (as indicated) were analyzed by RT-PCR ( n = 4; P = 0.0307). c The proline levels in the lung tissues were analyzed as described in the Methods ( n = 8 mice; P < 0.0001). d Collagen matrix was analyzed by SHG with multiphoton microscopy (top; bar, 100μm), immunostaining with antibody for collagen1A1 (middle; bar, 20 μm), or Masson’s trichrome staining (bottom; bar, 20 μm). The boxed areas in the IHC staining were enlarged and shown in the upper right corner. Bottom panels, for each method (as indicated in the figure) the mean intensities of collagen matrix in the Kras fl/+ ; P1 fl/fl group were quantified and compared to those of the Kras fl/+ group (normalized to 1; n = 30 fields from 6 mice for each group; different mice in each group were coded with different colors; P < 0.0001). Data in a – d represent mean ± SEM. Statistical significance was calculated using two-tailed unpaired Student’s t -test, * P < 0.05; *** P < 0.001. Source data are provided as a Source Data file.

Article Snippet: Immunohistochemistry was performed using the MaxVision TM HRP-Polymer anti-Rabbit IHC Kit (MXB biotechnologies) with rabbit antibodies against PINCH-1 (Abcam, ab108609,1:800), PYCR1 (Proteintech,13108-1-AP,1:800), DRP1 (Proteintech,12957-1-AP,1:800), Ki67 (CST,12202P, 1:1000) or collagen 1A1 (Novus,NB600-408).

Techniques: Immunostaining, Immunohistochemistry, Staining, Reverse Transcription Polymerase Chain Reaction, Microscopy, Two Tailed Test