Lasersharp 2000

Immunostaining was conducted on isolated islets, fixed in 4% paraformaldehyde before being permeabilised using 0.1% Triton X-100 (Sigma). After blocking with 5% goat serum, islets were incubated overnight (4°C) with primary antibodies before incubation with secondary antibodies. Fluorescent staining was visualised using a laser-scanning confocal microscopy (BioRad) controlled by LaserSharp2000 (BioRad).
Antibodies used in this study were: rabbit FITC 495-conjugated anti-GFP (1:250; DS-PB-00926; InsightBio, Wembley, United Kingdom), mouse anti-glucagon (1:1000; G2654; Sigma), guinea-pig anti-insulin (1:400; A0564; Dako, Santa Clara, California, United States), goat anti-somatostatin (1:100; sc-7819; Santa Cruz Biotechnology, Dallas, Texas, United States); Alexa Fluor 633 goat anti-mouse IgG (1:500; A-21052; ThermoFisher); Alexa Fluor 594 goat anti-guinea pig IgG (1:500; A-11076; ThermoFisher); and Alexa Fluor 546 donkey anti-goat IgG (1:100; A-11056; ThermoFisher).

TOPRO-3, ProLong Antifade kit, calcein, Alexa 546-conjugated dextran (M.W. 10,000), Alexa 594-conjugated-Tf, and non-essential amino acid solution (10 mM) were from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Lawrenceville, GA). DMEM was from Mediatech (Herndon, VA) or Sigma Aldrich (St. Louis, MO). Paraformaldehyde was from Fisher Scientific (Pittsburgh, PA). HEPES, Antibiotics, Chelex resin, ascorbate, ferric chloride, hemin and 2,2'-dipyridyl (DPD) were from Sigma Aldrich. All other chemicals of analytical grade were from Sigma Aldrich. Anti-DMT1 antibody (iron-responsive element, IRE) was described previously.^{(14 (link))} The Caco-2 cell line (HTB-37) was from American Type Culture Collection (Rockville, MD). The Millicell electrical resistance system and Millicell 6-well cell culture inserts with 0.4 µm pore size membranes were from Millipore (Bedford, MA). Transwell clear inserts for 24-well plates were from Corning (Lowell, MA). The collagen solution (rat tail Type I) was from Boehringer-Manheim (Manheim, Germany) and Becton Dickinson (San Diego, CA). DRAQ5 was from AXXOLA (San Diego, CA). The Bio-Rad Radiance 2000 Laser scanning microscope and Lasersharp 2000 were from Bio-Rad (Hercules, CA). The LSM 510 META Confocal microscope was from Carl-Zeiss (Thornwood, NY). Glass-bottom dishes were from Mat-Tek (Ashland, MA).

Embryos were dechorionated and fixed for 20 minutes in 10% formaldehyde overlayed with n-heptane at room temperature. Devitellinisation was achieved by adding chilled methanol and shaking vigorously. Embryos were washed twice with methanol before rehydrating in a step-wise manner with PBS and blocked with 4% normal goat serum. Imaginal discs from third instar larvae were fixed in 4% formaldehyde for 20 minutes before blocking.
Immunostaining of embryos was performed over night with anti-H B (directed against C-terminal fragment of H) and of wing discs with anti-H A (directed against central H fragment)22 (link), with anti-Phospho-Histone H3 (Ser28) (#9713, Cell Signaling Technology, Massachusetts, USA), and anti-GFP (sc-8334, Santa Cruz Biotechnology, Inc., Texas, USA). Secondary antibodies fused to FITC, Cy3 and Cy5 respectively were used (Jackson ImmunoResearch, Pennsylvania, USA). Embryos and imaginal tissue were mounted in VectaShield (Vector Laboratories, California, USA) before being imaged on a Bio-Rad MRC1024 system running LaserSharp 2000 imaging software (Bio-Rad Laboratories, California, USA) coupled to a Zeiss Axioskop (Carl Zeiss AG, Oberkochen, Germany). Figures were assembled using Corel Draw and Photo Paint software.

Antibody-labeled muscles were pinned down on Sylgard-bottomed petri dishes containing PBS and placed on the stage of an upright microscope (BX51WI, Olympus, Japan) equipped with a tunable Chameleon Ti/Sapphire laser (Coherent) and a Radiance 2000 Scanning Head (Bio-Rad, UK). Images were acquired with a ×20, 0.95 NA (Olympus XLUMPLANFL) water-immersion objective. Muscles labeled with Alexa 488 alone or Alexa 488 and Texas Red were excited with a laser wavelength of 840 nm. Images were simultaneously acquired using the following filter combinations (band-pass/dichroic/band-pass, in nm): second harmonic generation (SHG) and Alexa 488 emission (410-490//495//500-530), Alexa 488 and Texas Red emission (500-530//550//620-660), and SHG and Texas Red emission (402-446//495//615-645). Images were analyzed using commercial (LaserSharp 2000, Bio-Rad, UK) and/or public-domain image analysis software package (ImageJ).