Effects of fixative and fixation time for quantitative computerized image analysis of immunohistochemical staining

Fixation time plays a more critical role in quantitative computerized immun ohistochemistry than in routine qualitative studies. This study assessed th e significance of fixation time in methanol-Carnoy solution (MC) and 10% ne utral buffered formalin (NBF) relative to computerized quantitation of immu nohistochemical staining with 2 different antibodies. Normal skin specimens were fixed in NBF or MC solution for times ranging fr om 4 hr to 1 week. Paraffin sections were stained immunohistochemically wit h a polyclonal antibody for transforming growth factor-beta (TGF-beta) or a monoclonal antibody for thrombomodulin (TM). Extracellular matrix-associat ed TGF-beta immunoreactivity and TM-positive vessels were identified and as sessed with quantitative computerized image analysis. For both fixatives and antibodies, staining was superior in specimens fixed for 24 in. TGF-beta and TM stainings were stronger and more specific in MC -fixed tissues than in NBF-fixed tissues. We concluded that 24 hr in NBF an d MC is the optimal Fixation time for quantitative image analysis. MC is su perior to NBF for immunohistochemical staining of TGF-beta and TM.