Dp26 digital camera

Sections were deparaffinized and rehydrated in a series of ethanol. To reveal antigens, sections were heated to 97°C in citrate buffer. Sections were washed in TRIS‐Buffered Saline (TBS) (20 mmol/L Tris·HCl [Sigma], 137 mmol/L NaCl [Sigma], pH 7.6), and a solution of H₂O₂ in TBS was used to block endogenous peroxidase enzymes. Sections were rinsed in TBST (0.05% Tween‐20 in TBS), blocked in TBST+5% milk, and incubated overnight at 4°C with primary antibody (monoclonal rat‐anti‐CD45 antibody, 1:100, catalogue number 550539, BD Pharmingen, San Diego, CA) dissolved in TBST with 5% milk. Sections were rinsed in TBST and incubated 1 hour at RT with HRP‐conjugated secondary antibodies (polyclonal goat‐anti‐rat and anti‐rabbit antibodies, 1:1000, DakoCytomation) dissolved in TBST with 5% skimmed milk. Sections were rinsed in TBST before binding of antibody was visualized using 3,3′‐diaminobenzidine tetrahydrochloride hydrate solution (Sigma, D5637). Sections were counterstained with Mayer's hematoxylin, dehydrated, and mounted using Aquatex. Sections were analyzed with light microscopy using an Olympus BX51 microscope. Pictures were obtained using an Olympus DP26 digital camera.

A representative sample from the middle region of the colon was fixed in 4% paraformaldehyde at 4˚C for 24 h, embedded in paraffin, sectioned (5-µm), stained with hematoxylin (at room temperature for 5 min) and eosin (at room temperature for 2 min) and then observed using an IX 51 epifluorescence Olympus microscope (Olympus Corporation) equipped with a DP-26 digital camera at x40 magnification.