PRESERVATION OF TRYPAN BLUE AND NEUTRAL RED WITHIN THE CELLS OF LOOSE CONNECTIVE TISSUE

THEODORE SNOOK, Department of Anatomy, Syracuse University College of Medicine, Syracuse, N . Y .

A B S T R A C T . - T ~ ~ blue granules within the histiocytes and neutral red stained granules within the mast cells of the same areolar tissue spread can be preserved in their original colored state by the following method: fix in 10% formalin for 12-24 hours; rinse in dis- tilled water; place in two changes of dioxan for 5-10 minutes each (overnight in second change will not hurt); mount from the second dioxan using diaphane dissolved in dioxan as a mounting medium, or clear in xylene and mount in balsam. No dye is lost, and no cellular distortion occurs. Fast green is used as a counterstain (1% aqueous plus 0.2% acetic acid). Stain $ to 1 minute and rinse.

INTRODUCTION This method was devised to make permanent preparations of vi-

tally stained areolar tissue spreads, illustrating both intravital stain- ing by trypan blue and supravital staining by neutral red upon a single slide. A survey of the literature shows that various methods have been employed to preserve each of these dyes when. used separately. A critical analysis of the methods used for the fixation of trypan blue was given by Hetherington and Tompkins (1933). They claim that 10% formalin, as used by many investigators, is unsatisfactory when minute amounts of dye must be detected, altho it preserves the cell contours and type of dye deposition. They found that the fixing fluid of Carnoy and Lebrun gave perfect pres- ervation of the dye, but crystals of mercuric chloride caused some interference. Baird (1935) found that 5% neutral formalin fol- lowed by acetone dehydration satisfactorily preserved trypan blue in subcutaneous tissue spreads.

A survey of methods used was given by Cunningham and Conn (1932). Following the example set by McJunkin (1995), Zenker-formol has been used extensively as a fixative for neutral red and similar dyes. The problem of dehydration without the use of alcohols was solved by McJunkin and others by employing acetone, followed by benzene or xylene. Nu (1931) found that 100% alcohol, to which HgClz and neutral red had been added, permitted complete dehydration without any loss of neutral red from the cells. This method had the further

STIIV TECBNOLOOY. Tot. 14, So. 4, &OBER, 1939

Neutral red is more difficult to preserve satisfactorily.

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advantage that paraffin scctions of tissue vitally stained by ncutral red could he easily counterstained if the dye solution also containcd HF<’I, and nrutral red. Morrison, Gardner, and Hcevrs ( 1936) presrrvcd neutral rcd in I)araffin sections of gast ric mucosa by “Susa” fixation, followcd by 90”; and 100q; a h h d , clearing in carbon disnl- phide, and paraffin emhcylding. Whcn films can be ciritd, no dehydration p r o l h n is encountercd.

PROCEDTRE JIcdifications of the foregoing methtds were tried in order to prib-

serve both trypan bluc. and neutral red in the same spread o f tissue in as nrarly as possible their original appearance in the freshly made spread. Various counterstains were tried in order to bring out the

. . .. . FIG. 1. Identical wlls from an areolar tissue spread from a vitally stained white

rat, Iwfore fixation (.4), and after fixation, dehydration. and mounting (R) . 11, mast cells (neutral red); HI, histiocyte showing trypan blue granules: 119 histiiwyte with engorged Ieumyte. x 968.

fibrocytes and other tissue elements without altering the vital staining of the histiocytes and mast cells.

A single subcutaneous injection of 9.5 to 3 cc. of a fresh sterile lck, aqueous solution of vital trypan blue (Coleman and Bc4 Co.) for rats weighing 85-100 g. (5 cc. for mature rats) was sufficicbnt t o mark the histiocytes 48 hours after injection. Following light etherization, the animals were killed by severing a large artery. The neutral red solution was injected directly into the subcutaneous areolar tissues of the groin, the needle being directed so as to make several dye deposits around the original puncture. The sample used was certified WX 5) neutral red, Ehrlich. (79% dye content, S a - tional Aniline) 0.02% in 0.9% NaCI. In 5-5 minutes an incision was made and small blobs of the edematous tissue were reniovetl,

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TRTPAS BLUE ASD SEI'TRAI. RED IS COSSECTIVE TISSUE 141

placrd on clean slidcs, and teased into thin spreads by the use of necdlcs antl small pieces of filter paper. When the corners were dry m d attached to the slide, the spread was ready for fresh observation or for fixation.

The most brilliant results were obtained when 10% formalin was u s c d as the fisative. The slight acidity of the commercial formalin is dcsirahlc. because neutral red acts as an indicator, being red in acid antl orange or yellow in alkaline solutions. The 10% formalin should t w tested by adding to it a drop or two of neutral red and observing any color change. If it turns orange, cautiously add S 10 HCI until it turns red. Fixation is complete in a few hours, but it has been my practice to Ict the slides fix overnight. Several days fixation in formalin will not hurt the dyes.

FIG. 4. Permanent areolar tissue spread from white rat intravitally stained by trypan Iilue, supravitally stained by neutral red, and counterstaind tiy fast green. 21, mast cells (neutral red); H, histiocytes (trypan t h e ) ; F, filirocytt-s (fast green). x 268.

The slitles are first rinsed in distilled water and then placed in two successive changes of diosan (Coplin jars 1 and S), 3-5 minutes each depending on the thickness of the spread and the amount o f agitation. They may stay in the second change indefinitely. The slides are mounted directly from diosan, using as a medium hardcned diaphane (Will Corp.) redissolved in diosan. They may also hc placed in sylene and mountcd in halsam. The blue granules within the histio- cytes (macrophages) and the fine red granules of the mast cells have very near the same color values as they had in the fresh sprcads.

Zenker-formol (85 parts stock plus 15 parts formalin) also prcwrves the dyes well, altho they will h a w slightly different color values. HgC& crystals will br prcsent to somc dcgree. but 24 hours in diosan will rrmove a large proportion o f them.

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1 re STAIN TECHSOLOGY

The most useful counterstain tried was fast green F. C. F. (War- ner-Jenkinson Co., dye content 85%). A 1% solution in distilled water plus 0.2% glacial acetic acid for x-1 minute following the rinse after fixation brings out the nuclei, cytoplasmic processes, fibrocytes and fibers. After staining, rinse in distilled water and place in dioxan.

A few drops (M cc.) of Delafield’s hematoxylin added to 100 cc. of the 10% formalin fixing fluid stains the nuclei of the spread in 94 hours without interfering with the vital staining. The cytoplasm and fibers of the spread can then be stained by the use of 0.5% aqueous erythrosin (cert. no. CEr-52 Coleman and Bell) for 1 minute.

See fig. 9.

SUMMARY

By the above methods there is no appreciable loss of either trypan blue or neutral red following fixation, clearing, and mounting. Figure 1, A and B, shows the same cells before fixation, and after mounting. The three mast cells (M) have retained their relative positions, but the histiocytes have migrated, and also do not appear in the same focal plane in fig. 1, B that they had in fig. 1, A. By com- parison of the trypan blue granules of histiocyte 1 (HI) in each figure, it is evident that all of the dye is retained.

Slides made four years ago by essentially the same process have retained both dyes without any fading.

The most useful technic employing a counterstain is as follows

1. 52. 3. 4. 5. Dioxan l., 5 minutes. 6. Dioxan 9., 5 minutes. 7.

(see fig. 5 2 ) : Fixation-lO~o commercial formalin, 19-24 hours. Rinse in distilled water, 1-52 minutes. 1% fast green plus 0.!2yo glacial acetic acid, s - 1 minute. Rinse in distilled water, 1 minute.

Mount in diaphane dissolved in dioxan.

REFERENCES BAIED, T. T. Methylene blue and acid fuchsin for subcutaneous tissue spreads.

CUNNXNQIIA~ R. S. and CONN, H. J. Methods for the preservation of supra- vitally stained material.

HETEEEINGTON, D. C. and TOMPKINS, E. H. The fixation of tissues vitally stained with trypan blue. Stain Techn., 8, 31-4.

En, G. H. 1931. Technique for the complete preservation of supravital stain of neutral red in para511 sections. Proc. Soc. Exp. Biol. & Med., 29,258-9.

M ~ U N K I N , F. A. 1W. The origin of the mononuclear phagocytes of peritoneal exudates. Amer. J. Path., 1, SO5-24.

&ORRIBON, S., GARDNER. R. E. and REEVES, D. L. 19%. The selective elimination of neutral red through gastric mucosa. J. Lab. & Clin. Med., 21,839-7.

1935. Stain Techn., lo,%.

19S2. Stain Techn., 7, 1159.

19%.

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