Journal: Bone

Article Title: Contact of myeloma cells induces a characteristic transcriptome signature in skeletal precursor cells -Implications for myeloma bone disease.

doi: 10.1016/j.bone.2016.08.006

Figure Lengend Snippet: Fig. 4. ANGPTL4 expression in MSC after co-culture with INA-6 cells and CD19+ B-cells. Levels of ANGPTL4 mRNA in MSC after 24 h of (A) direct and indirect co-culture with INA-6 cells and (B) direct co-culture with CD19+ B-cells (n = 3). In case of direct co-culture, INA-6 and B-cells were depleted from MSC by MACS using CD45 and CD19 MicroBeads, respectively. Respective controls were treated accordingly. Expression of ANGPTL4 was determined with semi-quantitative RT-PCR by using densitometric analysis and normalization to levels of EEF1A1 mRNA. Bar graphs display the mean transcript levels ±SEM. Statistic was performed with non-parametric Kruskal-Wallis and Dunn's post-hoc multiple comparison tests (Fig. 4A) as well as two-tailed Mann-Whitney test (Fig. 4B). Differences were considered to be significant if the p-value was ≤0.05 (*).

Article Snippet: Briefly, white, clear-bottom 96- well cell culture plates (Greiner Bio-One GmbH, Frickenhausen, Germany) were washed once with 100 μl distilled H2O per well and supplemented with the solution of recombinant ANGPTL4 protein (ANGPTL4 full-length, R&D systems) or 1% bovine serum albumin (BSA) (control; Sigma).

Techniques: Expressing, Co-Culture Assay, Quantitative RT-PCR, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Bone

Article Title: Contact of myeloma cells induces a characteristic transcriptome signature in skeletal precursor cells -Implications for myeloma bone disease.

doi: 10.1016/j.bone.2016.08.006

Figure Lengend Snippet: Fig. 5. Adhesion of INA-6 cells to ANGPTL4 coating. INA-6 cells were pre-starved for 1 h and seeded on ANGPTL4-coated wells. After 1 h of incubation and plate washing, number of adherent INA-6 cells was determined using a luminescence-based assay. Percentage of attached cells was calculated by assessing cells attached at control wells (coated with 1% BSA) at 100%. Bar graphs display the means ± SEM of nine values (three experiments, triple values).

Article Snippet: Briefly, white, clear-bottom 96- well cell culture plates (Greiner Bio-One GmbH, Frickenhausen, Germany) were washed once with 100 μl distilled H2O per well and supplemented with the solution of recombinant ANGPTL4 protein (ANGPTL4 full-length, R&D systems) or 1% bovine serum albumin (BSA) (control; Sigma).

Techniques: Incubation, Luminescence Assay, Control

Journal: Reproduction

Article Title: Culture conditions antagonize lineage-promoting signaling in the mouse blastocyst

doi: 10.1530/rep-20-0107

Figure Lengend Snippet: Figure 1 BSA antagonizes signaling by exogenous FGF4. (A) Experimental design for embryos shown and analyzed in this figure. Red = NANOG indicative of epiblast cell fate, green = SOX17 indicative of primitive endoderm cell fate. (B) Immunostaining for SOX17 to identify primitive endoderm (PE) cells and NANOG to identify epiblast (EPI) cells in embryos cultured for 66 hours starting from two-cell stage in KSOM with 1 mg/mL BSA or without BSA (Millipore-Sigma MR-121-D and MR-107-D, respectively), but with 1 mg/mL added PVA (Sigma 360627). n = number of embryos examined. P = Student’s t-test. n.s. = not significant (P > 0.2). (C) Quantification of the total inner cell mass (ICM) and trophectoderm (TE) cells in embryos cultured in indicated conditions and proportion of ICM cells contributing to PE and EPI in these embryos. Each symbol represents a single embryo, column = mean, and error bars = standard deviations. P = Student’s t-test. n.s. = not significant (P > 0.2). (D) Immunostaining for SOX17 and NANOG in embryos cultured in KSOM + 150 ng/mL FGF4 (R&D Systems 235-F4-025) and 1 µg/mL heparin (Sigma H3149) in the presence or absence of 1 mg/mL BSA. Each symbol represents a single embryo, column = mean, and error bars = s.d. P = Student’s t-test. n.s. = not significant (P > 0.2). (E) Quantification of lineage specification in embryos cultured in conditions indicated. Each symbol represents a single embryo, column = mean, and error bars = s.d. (F) Response of embryos cultured in KSOM without 1 mg/mL BSA to low dose of exogenous FGF4 and 1 µg/mL heparin. Each symbol represents a single embryo, column = mean, and error bars = s.d. P = Student’s t-test.

Article Snippet: = not significant (P > 0.2). (D) Immunostaining for SOX17 and NANOG in embryos cultured in KSOM + 150 ng/mL FGF4 (R&D Systems 235-F4-025) and 1 μg/mL heparin (Sigma H3149) in the presence or absence of 1 mg/mL BSA.

Techniques: Immunostaining, Cell Culture

Journal: Arthritis Research & Therapy

Article Title: Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis

doi: 10.1186/ar1796

Figure Lengend Snippet: Effects of passages, starvation, cytokines and growth factors on ADAM15 expression in cultured cells. The mRNA expression of ADAM15 was examined by RT-PCR at 25 cycles as described in Materials and methods. (a) Effect of passages on the mRNA expression of ADAM15 in rheumatoid arthritis (RA) synovial fibroblasts (SFs). Lanes 1 to 5 indicate passages 5, 6, 7, 8 and 9 of RA SFs. (b) Effect of starvation on the mRNA expression of ADAM15 in RA SFs. (c) Effect of tumor necrosis factor (TNF)-α (0, 0.1, 1 and 10 ng/ml), IL-1α (0, 0.1, 1 and 10 ng/ml) or transforming growth factor (TGF)-β (0, 0.1, 1 and 10 ng/ml) on the mRNA expression of ADAM15 in RA SFs after stimulation with these factors for 24 h. (d) Regulation of the mRNA expression of ADAM15 by vascular endothelial growth factor (VEGF) 165 in RA SFs and human umbilical vein endothelial cells (HUVECs). Cells were stimulated with VEGF 165 (0, 1, 10 and 50 ng/ml) for 24 h. Note that VEGF 165 enhances the expression of ADAM15 only in HUVECs.

Article Snippet: To exclude the possible involvement of VEGFR-1 in ADAM15 expression, RA SFs and HUVECs were stimulated with recombinant human placenta growth factor (PlGF; 1, 10 or 50 ng/ml; R&D Systems), which selectively binds to VEGFR-1 [ ].

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis

doi: 10.1186/ar1796

Figure Lengend Snippet: Effects of cytokines and growth factors on expression of vascular endothelial growth factor receptors (VEGFRs). The mRNA expression of the VEGFRs was examined by RT-PCR at 30 cycles as described in Materials and methods. (a) The expression of VEGFR-1, VEGFR-2 and neuropilin-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) of different passages and human umbilical vein endothelial cells (HUVECs). Lanes 1 to 5 correspond to RA SFs of passages 5, 6, 7, 8 and 9, respectively. (b) The mRNA expression of VEGFR-1, VEGFR-2 and neuropilin-1 in RA SFs after 24 h stimulation with tumor necrosis factor (TNF)-α (0, 0.1, 1, 10 and 50 ng/ml), IL-1α (0, 0.1, 1, 10 and 50 ng/ml) or transforming growth factor (TGF)-β (0, 0.01, 0.1, 1 and 10 ng/ml). Note that TNF-α selectively induces the mRNA expression of VEGFR-2 in RA SFs.

Article Snippet: To exclude the possible involvement of VEGFR-1 in ADAM15 expression, RA SFs and HUVECs were stimulated with recombinant human placenta growth factor (PlGF; 1, 10 or 50 ng/ml; R&D Systems), which selectively binds to VEGFR-1 [ ].

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Arthritis Research & Therapy

Article Title: Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis

doi: 10.1186/ar1796

Figure Lengend Snippet: Immunohistochemistry of ADAM15, vascular endothelial growth factor receptor (VEGFR)-2 and von Willebrand factor (vWF). (a-d) Rheumatoid arthritis (RA) synovial fibroblasts (SFs) and (e-h) human umbilical vein endothelial cells (HUVEC) were cultured on Lab-Tek II chamber slides and immunostained with antibodies against (a,e) ADAM15, (b,f) VEGFR-2 or (c,g) vWF or (d,h) non-immune mouse IgG as described in Materials and methods. Immunostaining of VEGFR-2 in RA SFs (b) was performed with RA SFs that were treated with 10 ng/ml TNF-α for 24 h prior to immunohistochemistry. Note that vWF is not immunostained in RA SFs (c) , but VEGFR-2 is expressed in those stimulated with TNF-α (b) . Scale bar, 25 μm.

Article Snippet: To exclude the possible involvement of VEGFR-1 in ADAM15 expression, RA SFs and HUVECs were stimulated with recombinant human placenta growth factor (PlGF; 1, 10 or 50 ng/ml; R&D Systems), which selectively binds to VEGFR-1 [ ].

Techniques: Immunohistochemistry, Cell Culture, Immunostaining

Journal: Developmental biology

Article Title: Role of BMP signaling during early development of the annelid Capitella teleta.

doi: 10.1016/j.ydbio.2021.06.011

Figure Lengend Snippet: Fig. 1. List of exogenous BMP4 treatments performed. Solid black line: Animals raised in ASW þ PS þ BMP4 protein. Blue dashed line: Animals raised in ASW þ PS. Blue tissue in stages 4–6 larvae: neural tissue.

Article Snippet: Ct-BMP2/4 protein (Kenny et al., 2014) has a 42% sequence identity and 58% sequence similarity with the recombinant zebrafish BMP4 protein (R&D Systems, cat. 1128-BM-010) used; the sequence alignment was performed by T-Coffee (Di Tommaso et al., 2011).

Techniques:

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 1. Flow cytometry sorting of the combinatorial Scl library. (A) Library expression was monitored by staining with a phycoerythrin- conjugated antibody binding to a primary anti-c-Myc antibody. The purple rectangle gate indicates cells with the highest expression. (B) The library was incubated with 5 nM LRP4, and the low-affinity library fraction was collected (purple triangle). (C) The low-affinity library was incubated with 650 nM LRP6, and the purple rectangle represents clones that bind to LRP6.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Flow Cytometry, Expressing, Staining, Binding Assay, Incubation, Clone Assay

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 2. Identification of affinity-reducing mutations. Heat maps demonstrating significantly enriched Scl variants in (A) LRP4LOW library compared to SclNAIVE library fractions; (B) LRP4LOWLRP6 library compared to LRP4LOW library fractions; (C) LRP4LOW library compared to SclNAIVE library fractions that overlap with the LRP4LOWLRP6 library. The heat maps present the log2 transformation of the ER (red scale bar on the right-hand side) and highlight single mutations that significantly (A) reduce the binding affinity to LRP4, (B) reduce the binding affinity to LRP4 and retain binding to LRP6, and (C) overlap in (A) and (B). The substituting amino acids are shown on the X-axis, and the substituted positions are shown on the Y-axis. Statistical significance was determined by a two-sided Poisson exact test and multi-test corrected by the Benjamini–Hochberg FDR.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Transformation Assay, Binding Assay

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 4. YSD binding of SclWT and the selected single-mutation variants to LRP4. Geometric mean fluorescence intensity (Geo MFI) is presented as a fold change. Recombinant yeast cells expressing SclWT or its variants were incubated with (A) 1 nM, (B) 10 nM, or (C) 50 nM soluble LRP4. The binding signal of each Scl variant was normalized first to the expression signal of the corresponding variant and then to the binding signal of SclWT at the respective LRP4 concentration. Each experiment was repeated at least three times, and the results are presented as means SD. Statistical significance was assessed using an unpaired, two-tailed Student’s t-test. *P ≤0.05; **P ≤0.01; ***P ≤0.001.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Binding Assay, Mutagenesis, Recombinant, Expressing, Incubation, Variant Assay, Concentration Assay, Two Tailed Test

Journal: FEBS letters

Article Title: Mapping the sclerostin-LRP4 binding interface identifies critical interaction hotspots in loops 1 and 3 of sclerostin.

doi: 10.1002/1873-3468.15033

Figure Lengend Snippet: Fig. 7. SPR analysis of binding of purified SclWT and Scl single-mutation variants to LRP4. SPR data showing binding of (A) SclWT, (B) SclK75Q, (C) SclK75E, and (D) SclV136D to 3 μg of immobilized LRP4 receptor. Different protein concentrations are represented by different colors.

Article Snippet: Binding of the soluble purified SclWT protein and the SclK75E, SclK75Q, and SclV136D variants to recombinant human LRP4 (R&D Systems) was determined using a ProteOn XPR36 (Bio-Rad).

Techniques: Binding Assay, Mutagenesis

Journal: American journal of respiratory cell and molecular biology

Article Title: Angiopoietin-like 4 Is a Critical Regulator of Fibroblasts during Pulmonary Fibrosis Development.

doi: 10.1165/rcmb.2022-0304OC

Figure Lengend Snippet: Figure 1. Lung fibroblasts from fibrotic areas have a higher expression of ANGPTL4 (angiopoietin-like 4) than fibroblasts from nonfibrotic areas. (A) Representative images of a chest computed tomography scan, lung autopsy sample, and cultured lung fibroblasts isolated from nonfibrotic (NF) and fibrotic (F) areas of noncancerous regions of a patient with lung cancer with idiopathic pulmonary fibrosis (IPF) who underwent curative surgical resection. (B) Volcano plot of differential gene expressions by DNA microarray between fibroblasts derived from NF and F areas. Each point represents the average value of one transcript from three patients with IPF. Broken horizontal line represents a P value of 0.05. (C) ANGPTL4 mRNA levels in fibroblasts derived from NF and F areas among eight patients with IPF. Bars connecting points represent differences in individual patients. (D) Lung sections from a patient with IPF subjected to Masson’s trichrome staining, and immunohistochemical staining for anti-ANGPTL4 and anti–a-SMA. Scale bars: 1 mm; 200 mm. *P , 0.05. a-SMA = a-smooth muscle actin.

Article Snippet: In some experiments, recombinant ANGPTL4 (R&D Systems) at 1 μg/mouse was administered intranasally every other day from Day 3 after BLM administration.

Techniques: Expressing, Computed Tomography, Cell Culture, Isolation, Microarray, Derivative Assay, Staining, Immunohistochemical staining

Journal: American journal of respiratory cell and molecular biology

Article Title: Angiopoietin-like 4 Is a Critical Regulator of Fibroblasts during Pulmonary Fibrosis Development.

doi: 10.1165/rcmb.2022-0304OC

Figure Lengend Snippet: Figure 2. Angptl4 and fibrosis-related gene transcription is induced in the bleomycin (BLM)-induced pulmonary fibrosis (PF) model. (A) Lung sections from BLM-treated mice at Days 0, 3, 7, 14, and 21 (n = 5 each) were stained with Masson’s trichrome. Scale bars, 200 mm. (B) Expressions of Angptl4, Col1a1, and Col3a1 in BLM-treated mouse lungs. (C) Protein analysis of ANGPTL4 in BLM-treated mouse lungs. A representative blot is shown in the left panel and the quantified signal intensity of ANGPTL4 is shown in the right panel. GAPDH was used as an internal control. Data are shown as a box (median and 25th and 75th percentile) and whiskers (minimum to maximum) plot and are representative of two independent experiments. *P , 0.05, **P , 0.01, and ****P , 0.0001. MW = molecular weight markers; ns = not significant.

Article Snippet: In some experiments, recombinant ANGPTL4 (R&D Systems) at 1 μg/mouse was administered intranasally every other day from Day 3 after BLM administration.

Techniques: Staining, Control, Molecular Weight

Journal: American journal of respiratory cell and molecular biology

Article Title: Angiopoietin-like 4 Is a Critical Regulator of Fibroblasts during Pulmonary Fibrosis Development.

doi: 10.1165/rcmb.2022-0304OC

Figure Lengend Snippet: Figure 3. Single-cell RNA sequencing (scRNA-seq) of murine lung cells from normal and fibrotic lungs. (A) Fast interpolation-based t-distributed Stochastic Neighborhood Embedding (FItSNE) plots of all cells acquired by scRNA-seq for mouse lung samples. Cells were obtained from BLM- treated lungs from Days 0–21. (B) Gene expressions of Col1a1, Csf1r, and Angptl4 shown on FItSNE plots. Red to gray colors indicate high to low expression, respectively. (C) Violin plots showing gene expression levels of Col1a1, Acta2, and Angptl4 in lung fibroblasts at Days 0, 3, 7, 10, 14, and 21 after BLM treatment of lungs. (D) Percentage of Angptl4 and Col1a1-positive cells and the fold-change of the average

Article Snippet: In some experiments, recombinant ANGPTL4 (R&D Systems) at 1 μg/mouse was administered intranasally every other day from Day 3 after BLM administration.

Techniques: RNA Sequencing, Expressing, Gene Expression

Journal: American journal of respiratory cell and molecular biology

Article Title: Angiopoietin-like 4 Is a Critical Regulator of Fibroblasts during Pulmonary Fibrosis Development.

doi: 10.1165/rcmb.2022-0304OC

Figure Lengend Snippet: Figure 4. Expression of ANGPTL4 is regulated by TGF-b (transforming growth factor-b) signaling. (A) Expressions of ANGPTL4, ACTA2, COL1A2, and COL3A1 mRNA in normal human lung fibroblasts (NHLFs) stimulated with 5 ng/ml TGF-b for 0, 1, 2, 4, 8, and 24 hours. (B–D) Expression of ANGPTL4 mRNA (B), ANGPTL4 protein (C), ACTA2, COL1A2, and COL3A1 mRNA (D) in NHLFs stimulated with or without 5 ng/ml TGF-b for 2 hours (B and C) or 24 hours (D) after treatment with DMSO, SB, or U0126. Data are shown as the mean 6 SEM with an individual plot from three independent experiments. ***P , 0.001 and ****P , 0.0001. SB = SB431542.

Article Snippet: In some experiments, recombinant ANGPTL4 (R&D Systems) at 1 μg/mouse was administered intranasally every other day from Day 3 after BLM administration.

Techniques: Expressing

Journal: American journal of respiratory cell and molecular biology

Article Title: Angiopoietin-like 4 Is a Critical Regulator of Fibroblasts during Pulmonary Fibrosis Development.

doi: 10.1165/rcmb.2022-0304OC

Figure Lengend Snippet: Figure 5. Effect of ANGPTL4 on the fibroblast functions. (A) Expressions of ANGPTL4, ACTA2, COL1A2, and COL3A1 mRNA in NHLFs stimulated with or without TGF-b for 2 hours (ANGPTL4) and 24 hours (ACTA2, COL1A2, and COL3A1) after treatment with nontargeting siRNA (Ctrl) or siRNA for ANGPTL4. (B) A scratch was created with a pipette tip in NHLFs transfected with Ctrl siRNA or ANGPTL4 siRNA, and then cells were stimulated by TGF-b for 24 hours. Cell migration was evaluated by measuring areas between each edge of the scratch at 0 and 24 hours. Representative images are shown in the left panel and the quantified graph is shown in the right panel. Scale bars, 1 mm. (C) NHLFs transfected with Ctrl siRNA or ANGPTL4 siRNA were stimulated by TGF-b for 24 hours, and then cell proliferation was examined by MTT assay. Data are shown as mean 6 SEM with an individual plot from representative data of two independent experiments. *P , 0.05, **P , 0.01, ***P , 0.001, and ****P , 0.0001.

Article Snippet: In some experiments, recombinant ANGPTL4 (R&D Systems) at 1 μg/mouse was administered intranasally every other day from Day 3 after BLM administration.

Techniques: Transferring, Transfection, Migration, MTT Assay

Journal: American journal of respiratory cell and molecular biology

Article Title: Angiopoietin-like 4 Is a Critical Regulator of Fibroblasts during Pulmonary Fibrosis Development.

doi: 10.1165/rcmb.2022-0304OC

Figure Lengend Snippet: Figure 6. Intranasal administration of ANGPTL4 exacerbates BLM-induced PF. (A) Time-course schematic of the experiment. Recombinant (r) ANGPTL4 protein was administered intranasally to saline- or BLM-treated mice every other day from Day 3, and then the mice were euthanized on Day 14. (B) Representative microscopy images of Masson’s trichrome-stained lungs from PBS- or rANGPTL4-treated mice on Day 14 after saline or BLM administration. Scale bars, 200 mm. (C) Ashcroft scores indicating the level of fibrosis. (D and E) Hydroxyproline (collagen content) (D) and gene expressions of Col1a1 and Col3a1 (E) in PBS- or rANGPTL4-treated mice after saline or BLM administration. Saline i.t.- PBS i.n., n = 5; saline i.t.-rANGPTL4 i.n., n = 5; BLM i.t.-PBS i.n., n = 9; BLM i.t.-rANGPTL4 i.n., n = 9. Data are shown as a box (median and 25th and 75th percentile) and whiskers (minimum to maximum) plot and are representative of two independent experiments. *P , 0.05, **P , 0.01, and ***P , 0.001.

Article Snippet: In some experiments, recombinant ANGPTL4 (R&D Systems) at 1 μg/mouse was administered intranasally every other day from Day 3 after BLM administration.

Techniques: Recombinant, Saline, Microscopy, Staining

Journal: American journal of respiratory cell and molecular biology

Article Title: Angiopoietin-like 4 Is a Critical Regulator of Fibroblasts during Pulmonary Fibrosis Development.

doi: 10.1165/rcmb.2022-0304OC

Figure Lengend Snippet: Figure 7. PF induced by BLM treatment is attenuated in Angptl4 knockout (KO) mice. (A) Protein analysis of ANGPTL4 protein in BLM-treated lungs from wild-type (WT) and Angptl4 KO mice at Day 14. GAPDH was used as an internal control. (B) Representative microscopy images of Masson’s trichrome-stained lungs and in situ hybridization of Angptl4 in WT and Angptl4 KO mice at 14 days after saline or BLM treatment. Scale bars, 200 mm. (C) Ashcroft scores indicating the level of fibrosis. (D and E) Hydroxyproline (collagen content) (D) and gene expressions of Col1a1 and Col3a1 (E) at 14 days after saline or BLM treatment between WT and Angptl4 KO mice. WT mice (saline-treated, n = 4; BLM- treated, n = 8) and Angptl4 KO mice (saline-treated, n = 4; BLM-treated, n = 8). Data are shown as a box (median and 25th and 75th percentile) and whiskers (minimum to maximum) plot and are representative of two independent experiments. *P , 0.05 and **P , 0.01.

Article Snippet: In some experiments, recombinant ANGPTL4 (R&D Systems) at 1 μg/mouse was administered intranasally every other day from Day 3 after BLM administration.

Techniques: Knock-Out, Control, Microscopy, Staining, In Situ Hybridization, Saline