Serial sections of liver were cut on the cryostat and collected on Superfrost/Plus coated slides (Fisher Scientific, Pittsburgh PA) and processed for immunocytochemistry for albumin, biotin, F 4/80, glial fibrillary acidic protein (GFAP), or the endothelial cell marker CD-34. Slides with tissue sections were rinsed in Tris buffer three times and blocked for 1 hour in 3% normal goat serum (InVitrogen, Carlsbad CA) (for albumin, F4/80, CD-34) or 3% normal rabbit serum (for biotin). Each primary antibody was tested parametrically, in dilutions of Tris buffer in blocking solution, to determine the optimal antibody concentration to be used. The albumin antibody (Bethyl Labs; Montgomery TX) labeled with FITC was used at 1:500; the biotin antibody (Abcam; Cambridge MA) labeled with FITC was used at 1:500; the macrophage (Kupffer cell) antibody F/4/80 (Serotec, Raleigh NC) was used at 1:1000; the GFAP antibody (Dako; Carpinteria CA) was used at 1:3000; and the CD-34 antibody (Vector Labs) was used at 1:100. Sections were exposed to primary antibodies at room temperature and in the dark, overnight (16–18hr). The following day, slides were rinsed in Tris buffer three times. The sections for F4/80 then were incubated for 2 hours with either Alexa 488 or Alexa 546 goat anti-rat IgG at 1:1000 (Invitrogen; Carlsbad CA), sections for GFAP were incubated with either Alexa 488 or Alexa 546 goat anti-rabbit, at 1:1000 and sections for CD-34 were incubated with Alexa 488 goat anti-mouse at 1:1000. Following incubation, slides were rinsed with Tris buffer and coverslipped with Vectashield anti-fade fluorescent mounting medium with DAPI; DAPI served as a fluorescent (ultraviolet – UV) stain for cell nuclei.
Four groups of NOD and db/db mice were studied: non-diabetic, diabetic with normal emptying, diabetic with delayed gastric emptying, and diabetic with delayed gastric emptying reversed by the HO1 inducer hemin. Whole-mount samples from stomach were labeled in triplicate with antisera against F4/80, HO1, and CD206 and macrophages were quantified in stacked confocal images. Markers for macrophage subtypes were measured by quantitative PCR.
For immunofluorescence staining of adipose tissues, epididymal adipose tissues were fixed with 4% paraformaldehyde, after which epididymal fat was removed and minced into small pieces (~2 to 3 mm). The tissue pieces were boiled in 10 mM citrate buffer for 15 min and treated with 0.3% Triton X-100 in Tris-buffered saline (TBS; 150 mM NaCl and 20 mM Tris-HCl (pH 8.0)) for 10 min. Then, the tissue pieces were blocked with TBS containing 10% normal goat serum for 1 h at room temperature. In some experiments, Hoechst 33342 (Dojindo) and BODIPY (Molecular Probes) were added to the blocking solution. The tissue pieces were incubated with a primary antibody overnight at 4°C and then washed and probed with Alexa-conjugated secondary reagents. For immunofluorescence staining of pancreas, paraffin sections of pancreas were deparaffinized and rehydrated. The sections were boiled in 10 mM citrate buffer for 15 min and treated with 0.3% Triton X-100 in TBS for 10 min. Then, the sections were blocked with TBS containing 10% normal goat serum for 1 h at room temperature. Thereafter, sections were incubated with blocking solution containing primary antibodies and Hoechst 33342 overnight at 4°C. Finally, the sections were washed and probed with Alexa-conjugated secondary reagents. Fluorescent images were taken with an A1 confocal microscope configured with a Ti-E inverted microscope (Nikon). The number of crown-like structures (CLS) was determined by counting the total number of CLS in a 1 mm2 visual field.