Cellsense software

Immunofluorescence staining was used to examine BiP and 8-OHDG expression. After perfusion, the brain tissue was fixed in 10% neutral buffered formalin (Sigma), embedded in paraffin and cut into 5 μm sections. Next, the samples were deparaffinized, hydrated and stained by standard methods. For immunofluorescence analysis, brain sections were de-paraffinized. Antigen retrieval was performed by heating in 10mM sodium citrate buffer (pH 6.0) for 10 minutes using a microwave. Specimens were blocked in 5% blocking solution for 1 hour at room temperature followed by incubation with primary antibody at 4°C overnight. The BiP and 8-OHDG antibody were diluted 1:1000. The secondary antibodies used here were Alexa Fluor 647 or Alexa Fluor 488 antibodies corresponding to the species of primary antibody (1:1000). Coverslips were then dried and mounted onto slides using permount. The microscopy was performed using a fluorescence microscope (BX51; Olympus, Hamburg, Germany). The images were recorded and then processed using Olympus CellSense software (version 1.41).

Histomorphological analyses were performed in order to correlate histomorphological parameters of methacarn fixed tissue with the mucus layer thickness of cryopreserved tissue. As the mucus layer thickness could not be measured in the jejunum of cryopreserved samples, histomorphological parameters were detected in colonal tissue samples only. In total, 15 vertically oriented crypts per section were analyzed. For each crypt, the crypt depth and crypt area were measured. Furthermore, the absolute number of goblet cells (total number of cells per crypt) and the relative number of goblet cells (goblet cells per 1mm basement membrane of crypts) were determined. Moreover, the absolute mucin staining area (total mucin staining area per crypt) and the relative mucin staining area (mucin staining area in % of total crypt area) were detected. According to Hedemann et al.^{23 (link)} all mucus cells (goblet cells and crypt secretory cells), their apical secretion as well as the mucus material present in the crypt lumen were taken into account for the determination of the mucin staining area. Histological analyses were conducted with a light microscope (BX 43, Olympus, Hamburg, Germany), which was equipped with a digital camera (DP72, Olympus) and an image analysis program (Cell Sense software, Olympus).