Immunohistochemical staining of goldfish brain and gastrointestinal tract samples was used to study the anatomical distribution of ghrelin. The immunohistochemistry study was carried out as previously described [39 (link)]. Briefly, the above described cryostat sections were washed twice in 0.1M PBS and incubated twice in PBS containing 0.2% Triton and 0.5% of skimmed milk powder (45 min at room temperature). After overnight incubation with primary monoclonal antibody (mouse anti human ghrelin 1:200; ab57222, Abcam, Cambridge, MA, USA), previously used in goldfish by Kerbel and Unniappan [13 (link)], sections were washed three times in 0.2% Triton PBS and subsequently incubated with rabbit antimouse Alexa Fluor 488 (1:400; Invitrogen Molecular Probes, Eugene, OR, USA) for 2 hours at room temperature. A separate set of negative control slides were only treated with the secondary antibody (S1 Fig ). After washing in PBS, slides were coverslipped with Vectashield containing DAPI and observed with an epifluorescence microscope (Olympus Provis, equipped with a DP71 digital camera). Imaging processing was conducted as described previously for ISH.
Ab57222
Manufactured by Abcam
Sourced in United States, Canada
Ab57222 is a primary antibody that recognizes the Cytokeratin 18 protein. Cytokeratin 18 is a type I intermediate filament protein expressed in simple and stratified epithelial cells. The antibody can be used for the identification and localization of Cytokeratin 18 in various cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab47383, by Abcam (1 mentions)
Ab9361, by Abcam (1 mentions)
Hpa005653, by Merck Group (1 mentions)
H 031 31, by Phoenix Pharmaceuticals (1 mentions)
Mab1417, by R&D Systems (1 mentions)
A0566, by Agilent Technologies (1 mentions)
Ab200997, by Abcam (1 mentions)
3 protocols using ab57222
1
Immunohistochemical Mapping of Ghrelin
2
Comprehensive Immunofluorescence Staining Protocol
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Immunofluorescence was performed as described previously (26) . Rabbit anti-insulin (20056,1:1,000; Immuno-Star), guinea pig anti-insulin (4011-01F,1:100; LINCO), rat anti-insulin (MAB1417, 1:500; R&D Systems), rabbit antiglucagon (20076,1:2,000; ImmunoStar), guinea pig antiglucagon (1:500; LINCO), rabbit anti-somatostatin (A0566 1:500, DakoCytomation; and HPA019472, 1:2,500, Sigma-Aldrich), rabbit anti-PP (AB939,1:500; Chemicon International), chicken anti-b-galactosidase (b-gal) (ab9361,1:1,000; Abcam), mouse and rabbit anti-ghrelin (ab57222, 1:100, Abcam; and H-031-31, 1:1,000, Phoenix Pharmaceuticals), rabbit anti-MafA (34) , rabbit anti-MafB (HPA005653,1:5,000; Sigma-Aldrich), goat anti-Pdx1 (ab47383,1:5,000; Abcam), mouse anti-Isl1/2 (39.4D5, 1:50; DSHB), mouse anti-Ngn3 (F25A1B3, 1:50; DSHB), mouse anti-Pax6 (PAX6-S, 1:20; DSHB), mouse anti-Nkx6.1 (F55A10-C, 1:200; DSHB), and rat anti-Ki67 (14-5698, 1:50; Invitrogen) were used as primary antibodies. Secondary antibodies coupled to Cy3, Cy2, or Cy5 against rabbit, rat, mouse, chicken, or guinea pig (Jackson ImmunoResearch Laboratories) were used. Fluorescence imaging was performed using a Leica confocal microscope, and image processing was performed using Adobe Photoshop software.
3
Co-localization of Digestive Enzymes and Gut Hormones
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To determine whether ghrelin and/or nesfatin-1 co-localize with the digestive enzymes (sucrase-isomaltose; aminopeptidase A; trypsin and lipoprotein lipase), sections of intestine were incubated as described above with a mixture of primary antibody against ghrelin (mouse anti-rat ghrelin, Catalog # ab57222, Abcam, Toronto, ON, Canada) or nesfatin-1 (mouse anti-rat nesfatin-1, Catalog # ALX-804-854-C100, Enzo Life Sciences, Brockville, ON, Canada) and primary antibody against one of the four studied digestive enzymes (rabbit anti-human sucrase-isomaltase, Catalog # ab98872; rabbit antihuman aminopeptidase A, Catalog # ab109775; rabbit anti-human trypsin, Catalog # ab200997; rabbit antihuman lipoprotein lipase, Catalog # ab137821; all from Abcam, Toronto, ON, Canada). Each digestive enzyme antibody was diluted 1:200, at room temperature. Since heterologous antibodies were used in this work, positive immunostaining or immunoreactivity (IR) is referred to as sucrase-isomaltase-like IR, aminopeptidase A-like IR, trypsin-like IR and lipoprotein lipase-like IR. All these antibodies were previously used in goldfish (Bertucci et al., 2017b; Blanco et al., 2017b) . Western blot analysis using total proteins of pejerrey intestine was conducted to confirm the specificity of each antibody (Supporting Information Fig. S1). All antibodies show bands at the expected protein mass.
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