Journal: Investigative Ophthalmology & Visual Science

Article Title: CGRP Released by Corneal Sensory Nerve Maintains Tear Secretion of the Lacrimal Gland

doi: 10.1167/iovs.65.4.30

Figure Lengend Snippet: Regulation of CGRP on the proliferation of lacrimal acinar cells. ( A , B ) Representative images of immunolabeling in capsaicin-treated mice with or without CGRP administration using anti-MIST1 with anti-CCNA2, anti-Ki67, anti-PCNA, and anti-CDK1 ( n = 6 per group).

Article Snippet: The sections were incubated overnight at 4°C with mouse α-Mist1 (SC80984; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit α-CCNA2 (18202-1-AP; Proteintech, Chicago, IL, USA), rabbit α-Ki67 (ab16667; Abcam), rabbit α-PCNA (10205-2-AP; Proteintech), or rabbit α-CDK1 (19532-1-AP; Proteintech).

Techniques: Immunolabeling

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Mist1 Expression Is Required for Paneth Cell Maturation

doi: 10.1016/j.jcmgh.2019.07.003

Figure Lengend Snippet: Loss of Mist1 –/– in small intestine does not alter secretory allocation. ( A ) Staining for sucrase-isomaltase (enterocytes), alcian blue (goblet cells), and lysozyme (Paneth cells). Scale bars equal 100 μm, 50 μm, and 50 μm, respectively. Black arrows point out alcian blue + Paneth cells. ( B ) Quantification of chromogranin (Cg) A + cells in crypt and villus compartments. n = 3 mice per group. Quantification of alcian blue + cells in crypt and villus compartments. n = 3 mice per group. ** P <.01. Quantification of lysozyme + cells per crypt. n = 3 mice per group. ( C ) mRNA expression of transcription factors associated with secretory cell lineage allocation in isolated jejunal crypts from Mist1 +/+ and Mist1 –/– mice. n = 3–6 mice per group.

Article Snippet: After blocking in 5% bovine serum albumin in PBS for 1 hour at RT, primary antibodies were diluted in 1% bovine serum albumin in PBS and applied as follows: Mist1 (1:200, cat. #sc-80984; Santa Cruz Biotechnology, Santa Cruz, CA) sucrase-isomaltase (1:100, cat. #sc-27603; Santa Cruz Biotechnology); muc2 (1:100, cat. #sc-15334; Santa Cruz Biotechnology); and lysozyme (1:100, cat. #sc-27958; Santa Cruz Biotechnology) for 1 hour at RT.

Techniques: Staining, Expressing, Isolation

Journal: Stem Cell Research & Therapy

Article Title: Organ-specific extracellular matrix directs trans-differentiation of mesenchymal stem cells and formation of salivary gland-like organoids in vivo

doi: 10.1186/s13287-022-02993-y

Figure Lengend Snippet: Histological analysis of SMG-ECM-treated cultures reveals that aggregates display an expression pattern different from monolayer cells. A Paraffin sections of cells from 14 day cultures (i.e., untreated BM-MSCs [Untreated], SMG-ECM-treated aggregates [Agg/SMG-ECM], and monolayer [Mono/SMG-ECM] cells) were prepared and stained with H&E or PAS. PAS-positive cells are identified with arrows in the figure. Bar: 50 µm. B Immunofluorescence staining of cells prepared as in A and rat SMG tissue (positive control) was used to evaluate the presence and localization of amylase, Aqp5, Mist1, Krt14, Cldn3, and Cldn10 protein. Staining with nonspecific isotype antibody was used as a negative control (not shown). Bar: 50 µm

Article Snippet: The primary and secondary antibodies used in these studies were cross-reactive with rat and included the following: mouse Cytokeratin 14 monoclonal IgG3 antibody (Invitrogen, cat. # LL002), rabbit Claudin 3 polyclonal antibody (Invitrogen, cat. # 34-1700, Rockford, IL), rabbit Aquaporin 5 polyclonal antibody (Invitrogen, cat. # PA5-99403, Rockford, IL), mouse MIST1 monoclonal IgG1 antibody (Santa Cruz Biotechnology, cat. # sc-80984, Santa Cruz, CA), rabbit Claudin 10 polyclonal antibody (ThermoFisher, cat. # 38-8400, Rockford, IL), rabbit Amylase polyclonal antibody (Millipore Sigma, cat. # A8273, Saint Louis, MO), rabbit IgG isotype (Invitrogen, cat. # RI238244, Rockford, IL), mouse IgG1 isotype (BD Biosciences, cat. # 550878, San Jose, CA), mouse IgG3 isotype (BD Biosciences, cat. # 55034, San Jose, CA), goat anti-rabbit IgG (H&L) Alexa Fluor 647 (Abcam, cat. # ab150079, Cambridge, MA), and goat anti-mouse IgG Alexa Fluor 647 (ThermoFisher, cat. # A-21236, Eugene, OR).

Techniques: Expressing, Staining, Immunofluorescence, Positive Control, Negative Control

Journal: Stem Cell Research & Therapy

Article Title: Organ-specific extracellular matrix directs trans-differentiation of mesenchymal stem cells and formation of salivary gland-like organoids in vivo

doi: 10.1186/s13287-022-02993-y

Figure Lengend Snippet:

Article Snippet: The primary and secondary antibodies used in these studies were cross-reactive with rat and included the following: mouse Cytokeratin 14 monoclonal IgG3 antibody (Invitrogen, cat. # LL002), rabbit Claudin 3 polyclonal antibody (Invitrogen, cat. # 34-1700, Rockford, IL), rabbit Aquaporin 5 polyclonal antibody (Invitrogen, cat. # PA5-99403, Rockford, IL), mouse MIST1 monoclonal IgG1 antibody (Santa Cruz Biotechnology, cat. # sc-80984, Santa Cruz, CA), rabbit Claudin 10 polyclonal antibody (ThermoFisher, cat. # 38-8400, Rockford, IL), rabbit Amylase polyclonal antibody (Millipore Sigma, cat. # A8273, Saint Louis, MO), rabbit IgG isotype (Invitrogen, cat. # RI238244, Rockford, IL), mouse IgG1 isotype (BD Biosciences, cat. # 550878, San Jose, CA), mouse IgG3 isotype (BD Biosciences, cat. # 55034, San Jose, CA), goat anti-rabbit IgG (H&L) Alexa Fluor 647 (Abcam, cat. # ab150079, Cambridge, MA), and goat anti-mouse IgG Alexa Fluor 647 (ThermoFisher, cat. # A-21236, Eugene, OR).

Techniques:

Journal: Journal of Anatomy

Article Title: Comparing development and regeneration in the submandibular gland highlights distinct mechanisms

doi: 10.1111/joa.13387

Figure Lengend Snippet: Sox9 expression in embryonic and adult submandibular glands. (a–a″) Immunofluorescence of Sox9 (pink) in the embryonic E15.5 submandibular glands. (a) The adjacent embryonic sublingual gland (outlined in dashed yellow), which displays a similar pattern of expression to the submandibular gland. Dotted boxes in (a) indicate the area magnified in a′ and a′. (a′) Submandibular gland main duct, outlined by dotted white lines. (a″) Arrow head indicates forming acini with high levels of Sox9 expression. Arrow indicated forming intercalated duct with lower levels of Sox9. (b–d) Immunofluorescence of Sox9 (pink) and Mist1 (green) in the adult submandibular gland during homeostasis (female mouse 6–10 weeks). DNA is shown in blue (DAPI). (b) DAPI, Sox9, Mist1. Red dotted lines outline small intercalated ducts. White dotted lines outline acini. Yellow dotted lines outline larger striated/granular ducts. (c) Nuclear Mist1 highlights acini cells. (d) Sox9 is highest in Mist1‐negative intercalated ducts. SMG, submandibular gland; SL; sublingual gland. Scale bars: 100 μm (a‐a″); 50 μm (b–d)

Article Snippet: Primary antibodies and dilutions were used as follows: anti‐Fgf10 (Rabbit) 1:500 (ABN44, Millipore); anti‐Sox9 (Rabbit) 1:300 (AB5535, Millipore) and anti‐Mist1 (mouse) 1:100 (sc‐98771, Santa Cruz Biotechnology) which was used with the TSA kit for signal amplification (PerkinElmer), anti‐beta‐Galactosidase (chicken) 1:500 (Ab9361 Abcam), anti‐Ecadherin (mouse) 1:100 (ab76055 Abcam), anti‐RFP (rat) 1:200 (5F8 Chromotek) and anti‐PCNA (mouse) 1:100 (Thermofisher Scientific).

Techniques: Expressing, Immunofluorescence

Journal: Journal of Anatomy

Article Title: Comparing development and regeneration in the submandibular gland highlights distinct mechanisms

doi: 10.1111/joa.13387

Figure Lengend Snippet: Degeneration of acinar cells after ligation and regeneration after deligation. (a) Experimental strategy to study submandibular gland regeneration after duct ligation. (b–g) Trichrome staining of an unoperated (b, e), ligated (c, f) and regenerating (d, g) submandibular gland. All mice were female 6–10 weeks old. (h–j) Immunofluorescence for the acinar cell marker Mist1 (green: nuclear stain) in an unoperated (h), ligated (i) and regenerating (j) submandibular gland. DNA is shown in blue (DAPI). Arrowheads in (i) indicate a few resistant acinar cells after ligation. Scale bars (e) and (h): 50 μm. Same scale in f,g,i,j

Article Snippet: Primary antibodies and dilutions were used as follows: anti‐Fgf10 (Rabbit) 1:500 (ABN44, Millipore); anti‐Sox9 (Rabbit) 1:300 (AB5535, Millipore) and anti‐Mist1 (mouse) 1:100 (sc‐98771, Santa Cruz Biotechnology) which was used with the TSA kit for signal amplification (PerkinElmer), anti‐beta‐Galactosidase (chicken) 1:500 (Ab9361 Abcam), anti‐Ecadherin (mouse) 1:100 (ab76055 Abcam), anti‐RFP (rat) 1:200 (5F8 Chromotek) and anti‐PCNA (mouse) 1:100 (Thermofisher Scientific).

Techniques: Ligation, Staining, Immunofluorescence, Marker

Journal: Journal of Anatomy

Article Title: Comparing development and regeneration in the submandibular gland highlights distinct mechanisms

doi: 10.1111/joa.13387

Figure Lengend Snippet: Ectopic expression of Sox9 in striated/granular duct cells. (a) Percentage of Sox9‐positive cells (Sox9 + /DAPI) in the control‐unoperated and regenerating submandibular gland. N = number of mice. (b) Percentage of total Sox9‐positive cells (low and high expression) referred to as Sox9 + total (Sox9 + total/DAPI) and percentage of highly expressing Sox9‐positive cells, referred to as Sox9 + high (Sox9 + high/DAPI) found in the striated/granular ducts of control and regenerating submandibular glands. *** p < 0.001. Error bars are s.e.m. (c, d) Immunofluorescence of Sox9 (pink) in the control (c), and regenerating (d) submandibular gland. Dotted yellow lines delineate the striated/granular ducts. (e–g) Immunofluorescence for Sox9 (red) and Mist1 (green) in the regenerating submandibular gland acini. Dotted white line in (e‐g) delineates a Mist1‐positive acinus. (e) Sox9 (red) overlaps with DAPI (Blue) in the nucleus. (f) Sox9. (g) Mist1. Arrowheads (e–g) point to acinar cells (Mist1 positive) with strong Sox9 expression. Arrows point to intercalated ducts with strong Sox9 expression but no Mist1. All mice were female 6–10 weeks old. Scale bar (c): 50 μm, same scale in (d). (e) 25 μm, same scale in (f, g)

Article Snippet: Primary antibodies and dilutions were used as follows: anti‐Fgf10 (Rabbit) 1:500 (ABN44, Millipore); anti‐Sox9 (Rabbit) 1:300 (AB5535, Millipore) and anti‐Mist1 (mouse) 1:100 (sc‐98771, Santa Cruz Biotechnology) which was used with the TSA kit for signal amplification (PerkinElmer), anti‐beta‐Galactosidase (chicken) 1:500 (Ab9361 Abcam), anti‐Ecadherin (mouse) 1:100 (ab76055 Abcam), anti‐RFP (rat) 1:200 (5F8 Chromotek) and anti‐PCNA (mouse) 1:100 (Thermofisher Scientific).

Techniques: Expressing, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Immediate and Late Effects of Early Weaning on Rat Gastric Cell Differentiation

doi: 10.3390/ijms21010196

Figure Lengend Snippet: Early weaning affects gastric cell distribution through transient and long- lasting effects on MNC, TC and ZC in the gastric mucosa of S and EW rats at 18 and 60 days. ( A ) Representative photomicrographs of the gastric mucosa illustrate the distribution of mucous neck cells (MNC: +GSII/FITC), transition cell (TC: +GSII/FITC+Mist1/Cy3 or +GSII/FITC +Mist1/Cy3+GIF/AlexaFluor 633) and zymogenic cells (ZC: +Mist1/Cy3). Nuclei were counterstained with DAPI. Representative images were acquired using confocal microscopy (Zeiss LSM 780-NLO). Scale bar = 50 μm. The insets show in detail the morphology and labeling of each cell type; digital zoom was set at 57%. ( B ) Expression of Muc6 after RT- qPCR represented as fold induction of internal control ( Actb ). n = 3–5 per group. Technical replicates: n = 2. ( C – E ) The quantification of each cell type/field/animal was performed for ( C ) as MNC/field/animal; ( D ) TC/field/animal after counting double- labeled (+GSII/FITC+Mist1/Cy3) or triple- labeled (+GSII/FITC+Mist1/Cy3+GIF/AlexaFluor 633) and ( E ) ZC/field/animal. To determine the density of each cell type, five fields/rat were used, considering the central area in corpus region and longitudinally sections glands. For quantification, images were acquired under fluorescence microscopy (Axioscope 2, Zeiss). Individual results are represented for S (blue) and EW (red) rats and means ± SD is indicated in each group and age. Unpaired Student t test was used and * p < 0.05, ** p < 0.01.

Article Snippet: Then, slides were washed in PBS, and goat anti-GIF (2 µg/mL) and monoclonal mouse anti-Mist1 (8 µg/mL) were added for incubation (4 °C, overnight) (sc 161643 and 80984, Santa Cruz Biotechnology).

Techniques: Confocal Microscopy, Labeling, Expressing, Quantitative RT-PCR, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Immediate and Late Effects of Early Weaning on Rat Gastric Cell Differentiation

doi: 10.3390/ijms21010196

Figure Lengend Snippet: Early weaning alters the expression of regulatory genes and protein levels in zymogenic cells in the rat gastric mucosa. ( A ) Expression of Bhlha15 , Gif , Pga5 , Pgc was compared in gastric mucosa of S and EW rats at 18 and 60 days and levels are indicated as fold induction of internal control ( Actb ). Biological replicates: n = 3–10 animals. Technical replicates: n = 2–3. ( B ) tissue lysates from gastric mucosa were used to detect the transcription factor Mist1 (22 kDa) and pepsinogen C (42 kDa) at 18, and 60 days. Protein levels were determined by integrate optical densitometry (arbitrary units) after immunoblots and compared to the internal control protein ( β -actin, 42 kDa). Individual results are represented for S (blue) and EW rats (red) and means ± SD is indicated in each group and age. After unpaired Student t test between groups/age: *** p < 0.001; * p < 0.05, ** p < 0.01.

Article Snippet: Then, slides were washed in PBS, and goat anti-GIF (2 µg/mL) and monoclonal mouse anti-Mist1 (8 µg/mL) were added for incubation (4 °C, overnight) (sc 161643 and 80984, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Immediate and Late Effects of Early Weaning on Rat Gastric Cell Differentiation

doi: 10.3390/ijms21010196

Figure Lengend Snippet: Summary of early weaning effects on the differentiation of gastric cells: transient and permanent responses. ( A ) Heat map of gastric mucosa gene expression data at different ages. Red represents increased gene expression, while green represents reduced gene expression. ( B ) Principal Component Analysis (PCA) of gastric mucosa gene expression data. PCA two-dimensional scatter plot represent the differential gene expression patterns of suckling and early-weaning rats at different ages. Axis: X = Dim1-Principal Component 1 (63.11% variance); Y = Dim2-Principal Component 2 (13.15% variance). ( C ) Representative images from the gastric mucosa of suckling and early- weanling rats at 18, and 60 days. Sections were used for multiple labeling to detect: mucous granules (GSII/FITC) in MNC and TC; Mist1/Cy3 in the nuclei of TC and ZC; GIF/AlexaFluor 633 in serous granules in TC and ZC. Nuclei were counterstained with DAPI. Images were obtained under fluorescence microscope, using separated filters, and they were combined through Zen Software. Acquisition at X 40. Bar = 50 μm. ( D ) Schematic distribution of PC, MNC, TC and ZC in the gastric mucosa (F—foveola; I + N—isthmus and neck; T—transition; B—base).

Article Snippet: Then, slides were washed in PBS, and goat anti-GIF (2 µg/mL) and monoclonal mouse anti-Mist1 (8 µg/mL) were added for incubation (4 °C, overnight) (sc 161643 and 80984, Santa Cruz Biotechnology).

Techniques: Expressing, Labeling, Fluorescence, Microscopy, Software

Journal: International Journal of Molecular Sciences

Article Title: Immediate and Late Effects of Early Weaning on Rat Gastric Cell Differentiation

doi: 10.3390/ijms21010196

Figure Lengend Snippet: Primers used for detection of gene expression after qPCR in ontogenic analyses.

Article Snippet: Then, slides were washed in PBS, and goat anti-GIF (2 µg/mL) and monoclonal mouse anti-Mist1 (8 µg/mL) were added for incubation (4 °C, overnight) (sc 161643 and 80984, Santa Cruz Biotechnology).

Techniques: Expressing