Immunofluorescence Microscopy of Cyanurated Tissues

Publisher Website
Google Scholar
Abstract
Test tissues consisted of: (1) popliteal lymph nodes of rabbits, removed 6 hr after injection of hind footpads with 0.2 ml of 125 mg/ml solution of 5× crystallized chicken ovalbumin, and (2) lungs from guinea pigs, passively sensitized with rabbit antiovalbumin serum, then anaphylactically shocked by intracardial injection of a 1% chicken ovalbumin solution. Similar control tissues from normal rabbits, and lungs of passively sensitized guinea pigs, but shocked with histamine instead of ovalbumin, were included. Pieces of fresh tissue not exceeding 2 mm3 were fixed as follows: (1) Cyanuration—lymph nodes, 1 hr; lung, 0.5 hr; both at 23–27 C—in anhydrous methanol containing 0.5% w/v cyanuric chloride and 1% v/v N, N-diethylaminoethanol. (2) Control fixatives—all specimens 18-24 hr at 4—6 C—absolute methanol; 95% ethanol; neutral buffered 10% formalin; and an FAA mixture (formalin, conc., 6; glacial acetic acid, 2; 30% ethanol, 92). Freeze-dried material was either left unfixed (a control) or fixed in xylene or toluene containing 0.5% w/'v cyanuric chloride and 1% v/v N, N-diisopropylaminoethanol; time and temperature as for fresh tissues. All tissues were routinely dehydrated, cleared, and vacuum embedded in an ester wax, diethylene glycol distearate, or in paraffin at 52 C. Sections 2-4 μ thick were attached to gelatin-coated slides, the wax removed in petroleum ether, and stained 20 min at 23-27 C in a 0.10% solution of fluorescein isothiocyanate-conjugated rabbit antiovalbumin globulin, washed in phosphate buffered saline 10 min, dehydrated, cleared and covered in a nonfluorescent medium. With ultraviolet illumination, brightly immunofluorescent, anti-genically specific staining was obtained in cyanurated fresh and freeze-dried lymph node and lung tissues. In contrast, specific staining was diminished or absent in comparable tissues reacted in the control fixatives.