Jun 28, 1983 - After touch impressions were made, the brains were divided, with ... that too little glue was as ineffective as no glue at all and that too much.
Vol. 18. No. 4
JOURNAL OF CLINICAL MICROBIOLOGY, OCt. 1983. p. 968-971 0095-1 137/83/100968-04$02.00/0 Copyright © 1983, American Society for Microbiology
NOTES Increased Immunofluorescent Staining of Rabies-Infected, Formalin-Fixed Brain Tissue After Pepsin and Trypsin Digestion FRANCES L. REID,'* NANCY H. HALL,- JEAN S. SMITH., AND GEORGE M. BAER' Viral and Rickettsial Zoonoses Branch, Diviision ot Viral Diseases, Ceniter for Infectious Disease, Centers for Disease Control (Lawrencev,ille Facilitxv), Lawrencev ille, Geor-gia 30246, andic University Hygienic Laboratory, University of lowa, Iowa City-, Iowa 522422 Received 11 April 1983/Accepted 28 June 1983
This study was undertaken to evaluate the sensitivity of the direct immunofluorescence test on Formalin-fixed, trypsin-digested, rabies-infected brain tissue. Our results suggest that the optimal unmasking of rabies antigenic sites is obtained by using a double enzyme digestion with pepsin and trypsin in lieu of only trypsin.
Immunofluorescent staining of rabies antigen in Formalin-fixed brain tissue was first reported by Johnson et al. (10). Until then, only fresh, fresh-frozen, or glycerine-preserved tissues could be used to immunologically identify rabies virus antigen. In reporting their findings, Johnson and his co-workers stressed the need for more study into the sensitivity of this method as compared with the direct immunofluorescence (IF) (4) and mouse inoculation (11) tests on unfixed tissue. When we first used the method of Johnson et al. (10), we noted a definite lack of sensitivity, and although a positive test could be considered conclusive because specificity controls were included, a negative test was inconclusive because of this poor sensitivity. In our attempt to make this procedure comparable to the direct IF and mouse inoculation tests, we made several modifications to the original procedure. The most significant change was tissue digestion with two enzymes, pepsin and trypsin. ICR mice were inoculated intracerebrally with 10 mouse intracerebral lethal doses (MICLD50) of MD5951 street rabies virus (obtained from Mario Martell, Mexico City, Mexico). Brains were collected each day beginning on day 3 postinoculation (symptoms generally appear by day 6) through day 13, after which all remaining mice had died. A total of 51 brains were collected. After touch impressions were made, the brains were divided, with one-half of each being placed in 10% buffered neutral Formalin. A 10% suspension in 0.75% bovine albumin diluent was made of the other half of each brain and stored at -70°C. The direct IF test was performed on the
impression slides made from fresh brain material, and the results were recorded as positive, weak positive, or negative. Serial 10-fold dilutions were made from the frozen brain suspensions, and 3-week-old mice were inoculated intracerebrally to determine the virus titer. Of the 51 brains, 42 were positive by both direct IF and mouse inoculation of fresh brain material. The Formalin-fixed mouse brains were blind coded and, after 10 days of fixation, processed in a routine histological manner and embedded in paraffin. Tissue sections were cut, mounted (two per slide) on Histostix (Accurate Chemical and Scientific Corp., Westbury, N.Y.)-coated slides, and dried for 24 h at 60°C. All slides were then deparaffinized in xylene, rehydrated through graded ethanol, and rinsed in Ca2+-Mg2+-free phosphate-buffered saline according to the method described by Johnson et al. (10). Four identical sets of slides (51 slides per set), plus positive and negative controls, were tested in the following manner. Set 1 was stained without enzyme digestion. Set 2 was stained after digestion in 0.25% trypsin (1 x trypsin; GIBCO, Grand Island, N.Y.) for 3 h. Set 3 was stained after digestion in 0.4% pepsin (Pepsin 1:60,000; Sigma Chemical Co., St. Louis, Mo.) for 1 h. Set 4 was stained after digestion in 0.4% pepsin for 1 h followed by digestion in 0.25% trypsin for 90 min.
All digestions were carried out at room temperature. After digestion, the slides were washed for 30 min in phosphate-buffered saline (0.01 M, pH 7.6). One tissue per slide was then stained with normal mouse brain-adsorbed rabies conjugate. The second tissue section on 968
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each slide was stained with rabies virus-adsorbed rabies conjugate. All slides were examined at x400, using a Zeiss Standard Universal microscope with vertical illumination. The light source was an XBO 150-W xenon bulb with KP490 and LP510 filters. In preliminary studies (data not shown), we discovered that the Formalin-fixed tissues often washed off the slides during digestion or staining. In attempts to solve this problem, we coated the slides with various amounts of Elmer's glue (Borden, Inc., Columbus, Ohio) before mounting the tissues. We found that too little glue was as ineffective as no glue at all and that too much glue gave an unacceptable amount of autofluorescence. Coating the slides with a 2% phenol-gelatin solution (7) resulted in loss of the tissues from the slides during testing. Precoating slides with Histostix solution, coupled with a 24h hot air (60°C) drying after mounting the tissue, proved effective in keeping the tissues on the slides even after prolonged enzyme digestion. In early trials, we also found that the test did not give consistently reproducible results. We varied the CaCl. concentration from 0.02% (10) to 0.1% (9), as suggested by Mepham et al. (12), and discovered that the higher concentration of calcium ions produced brighter fluorescence and more consistent results (data not shown). However, the immunofluorescent staining of Formalin-fixed tissue was still pale in comparison with the direct IF test on fresh brain tissue. To achieve a 4+ staining intensity, we had to increase the conjugate concentration fourfold over that used on fresh tissue. Concentrations of trypsin ranging from 0.25 to 2.5% were then tested at digestion times of 1, 2, 3, 4, and 5 h. TABLE 1. Comparison of immunofluorescent staining on 18 low-positive (
