In addition prolonged fixation in formalin caused a signal reduction for K-7, but did not affect routine HE and reticulin staining. The difference is most GANT61 in vivo likely due to changes in epitopes required for immunohistochemistry,

but less for routine HE and reticulin staining. Blebbistatin Indications for possible overfixation by formalin were present in K-7 and possibly in MRP2 staining. Signal reduction in K-7 stained biopsies was associated with increased fixation time and was also present in the periphery of wedge biopsies (24 hrs and 5 days fixation). In both situations, prolonged exposure to formalin could explain epitope masking due to protein cross linking of the tissues antigens. Consequently, this antigen masking could result in decreased antigen-antibody reactivity. Occurrence and intensity of this effect will vary per antibody as not all epitopes will be affected similarly [18]. Immunohistochemical reactivity was optimal after formalin fixation and replacement of the formalin by ethanol 70% within 1 – 4 hrs. Formalin fixation ABT-888 molecular weight proved necessary for assessment of copper accumulation in liver tissue. Routine rubeanic acid staining was sufficient in a wedge biopsy (24 hrs) as well as in a Menghini

biopsy (8 hrs). Reliable rhodanine staining was limited to a wedge biopsy only. RNAlater or Boonfix treated slides did not produce a sufficient signal in any of the investigated copper stains. Interestingly, previous exposure to

HCl damp in rubeanic acid staining, as was suggested to enhance copper staining [18], completely inhibited the signal in all slides and therefore proved to be ineffective. Conclusion Summarized, in the search to decrease the number of biopsies needed for molecular and (immuno)histochemical analysis, it turned out that at least two biopsies (10% neutral buffered formalin and RNAlater) are needed. Since both biopsies can be dispersed in relatively non-toxic liquid preservatives, this combination can easily provide researchers with material for SDHB high throughput expression analysis. Moreover it nicely resembles the sample preparation protocols that are commonly used in clinics today. Since biopsies fixed in either RNAlater or formalin remain stable at room temperature, transport is easy from the clinical situation to the research facility for further processing as well as prolonged storage. Results of our study showed that a reduction of the formalin fixation time to 1 to 4 hrs will generally reduce formalin induced reduced staining and staining artifacts. Therefore, any extension of the formalin fixation period should be discouraged when immunohistochemistry is considered. In view of the large similarities between human and canine liver diseases [19], it is conceivable that the protocols described here can be easily translated into the human biomedical field.