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Non-specificity of crystal violet staining for Renin granules
Acta histochem. Bd. 58, S.191-193 (1977)
Istituto di Anatomia e Istologia Patologica, Universita degli Studi di Modena, Italy
Non-specificity of crystal violet staining for Renin granules By GIUSEPPE BARBOLINI and ACHILLE OARLO PESSINA With one figure (Received March 29, 1976)
Summary This study deals with the histochemical specificity of crystal violet staining. Histological dyes such as crystal violet (CI 42555), ethyl violet (CI 42600) and methyl violet (CI 42535) are regularly employed for the assessment of juxtaglomerular granulation (WILSON 1952; FRIEDBERG and REID 1966; MARIUZZI and NENOI 1966). WILSON'S modification of BOWIE's procedure is the staining method most frequently used for demonstration of 'renin granules' in the juxtaglomerular cells (JC). It has been assumed that JC granularity parallels renin-induced hypertension (PITTS 1968).
Materials and Methods Tissue samples from rat and human kidney were fixed in BOUIN, ZENKER or formalin and embed· ded in paraffin wax. Alternatively they were freeze-dried in a thermoelectric tissue dryer, and fixed in formalin vapour at 70°C for 2 h, before embedding in paraffin wax (PEARSE 1968). Formalinfixed, paraffin·en,beddcd, sections of rat and hUll"lan tissues other than kidney were used as controls. In vitro tests were performed using purified angiotensinogen, angiotensin I, angiotensin II and renin!).
Results In sections of rat and human kidney not only the granules of the JO, but also red blood cells and some nerve fibres, were stained deep purple. In the sections from various control tissues, myelinated tracts in the brain (corpus callosum, stria medullaris), striated muscle (quadriceps, psoas and deltoid), sweat glands (Fig. 1) and the intercellular bridges of the epidermis were likewise stained. In vitro, WILSON'S crystal violet solution2 ), applied for 2 to 24 h, did not stain angiotensinogen, angiotensin I, angiotensin II or renin. 1) Rabbit serum renin-substrate (COOK et al. 1965), human serum renin-substrate (HAAS et al1966), angiotensin I (supplied by courtesy of Dr. TESSER, Department of Organic Chemistry, Uni. versity of Nijmegen), angiotensin II (hypertensin, CIBA), pig renin (PEART et al. 1966), rat renin (CUTHBERT et al. 1966). 2) 100 ml of 20 % ethyl alcohol to which has been added 10 drops of the stock solution of 1 % aqueous crystal violet. 13
Acta hi8tochcm. Bd. 58
192
G. BARBOLINI and A. C. PESSINA
Fig. 1. Human digital biopsy according to CURRI et a1. (1966): sweat glands. The secretion granules of the secretory segment (TISCHENDORF et a1. 1971) are stained deep purple by the crystal violet. X 1472.
In our opmlOn, WILSON'S crystal violet does not stain renin but probably its proteolipid carrier. Evidence that the modified crystal violet stain is able to demonstrate proteolipids is given in a separate paper.
Literature COOK, W. F., and LEE, M. R., The preparation of rabbit renin·substrate for the assay of minute amounts of renin . Biochem. J . 96,413-418 (1965). CURRI, S. B., MANZOLI, D., and TISCHENDORF, F. , Die Fingerbeerenbiopsie. Klin . Wschr. 44,584 - 590 (1966). CUTHBERT, M. F., ASSCHER, A. W., and HERY JONES, J., Characterisation of a vascular permeability factor of renal origin. Clin. Sci . 31, 325-336 (1966). FRIEDBERG, E. C., and REID, J. D., Rapid staining of the juxtaglomerular cell granules and renal tubular mitochondria for light microscopy. Nature (Lond.) 209, 1143-1144 (1966). HAAS, E., GOLDBLATT, H., GIPSON, E. E., and LA VERA LEWIS, B. S., Extraction, purification and assay of human renin free of angiotensinase. Clin. Res. 19, 739-749 (1966).
Acta
hi$to('h~m.
Plaie III
Ed. :j8
2
4
6
5
Wari ;;, H ec hardt and Parta nell VEB GUSTAV FISCHER VERLAG lENA
Acta hiatochem. Bd. 58
Plate IV
1
3a
3b
4
5
6
7
Kasabyan and Pisarev VEB GUSTAV FISCHER VERLAG lENA
Acta histochem. Ed. 58
Planche V
2
3
4
5
6
Strambi VEB GUSTAV FISCHER VERLAG JENA
Tafel VI
A ci(t hitslochem. tid. 58
1
2
3
4
5
Dunz e ndorfer und Web e r VEB GUSTAV FISCHER VERLAG lENA
Acta hi8tochern. Ed. 58
'l'afel VII
la
Passia, vVeissbach, Muller u. a.
Ib
T afel VIII
Acta histochem . Bd. 58
2a, b
l'assia. W e i ssbac h , Mull e r u . a.
Acta histochem. Bd. 58
'l'ajellX
3a-3c
Passia,
Wci~sbach,
Muller u. a.
Non-specificity of crystal violet staining
193
MARIUZZI, G_ M., and NEXCI, I., Nuova tecnica istologica per evidenziare Ie cellule granulose epitelioidi degli apparati juxtaglomerulari. Riv. Pat. Olin. Spero 7, 365-368 (1966). PEARSE, A. G. E., Histochemistry: theoretical and applied. 3rd ed. vol. 1, p. 51. J. & A. Ohurchill, London 1968. PEART, 'V. S., LLOYD, A. M., THATCHER, G. M., LEVER, A. F., PAYNE, N., and STONE, N., Purification of pig renin. Biochem. J. 99, 708-716 (1966). PITTS, R. F., Physiology of the kidney and body fluids. 2nd ed. p. 158. Year Book Med. Publ. Inc., Ohicago 1968. TISCHEXDORF, F., BARBOLINI, G., and OURRI, S. B., Histology, histochemistry and function of the human digital arteriovenous anastomoses. II. The possible relationship with sweat glands. }licrosc. Res. 3,323-336 (1971). WILSON, W., A new staining method for demonstrating the granules of the juxtaglomerular complex. Anat. Rec. 112,497-507 (1952). Address of the authors: Prof. Dr. GIUSEPPE BARBOLINI, Istituto di Anatomia e Istologia Pato10gica, Universita clegli Studi di Modena, Via Berengario, 4, I - 41100 Modena, Italia.
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Istituto di Anatomia e Istologia Patologica, Universita degli Studi di Modena, Italy
Non-specificity of crystal violet staining for Renin granules By GIUSEPPE BARBOLINI and ACHILLE OARLO PESSINA With one figure (Received March 29, 1976)
Summary This study deals with the histochemical specificity of crystal violet staining. Histological dyes such as crystal violet (CI 42555), ethyl violet (CI 42600) and methyl violet (CI 42535) are regularly employed for the assessment of juxtaglomerular granulation (WILSON 1952; FRIEDBERG and REID 1966; MARIUZZI and NENOI 1966). WILSON'S modification of BOWIE's procedure is the staining method most frequently used for demonstration of 'renin granules' in the juxtaglomerular cells (JC). It has been assumed that JC granularity parallels renin-induced hypertension (PITTS 1968).
Materials and Methods Tissue samples from rat and human kidney were fixed in BOUIN, ZENKER or formalin and embed· ded in paraffin wax. Alternatively they were freeze-dried in a thermoelectric tissue dryer, and fixed in formalin vapour at 70°C for 2 h, before embedding in paraffin wax (PEARSE 1968). Formalinfixed, paraffin·en,beddcd, sections of rat and hUll"lan tissues other than kidney were used as controls. In vitro tests were performed using purified angiotensinogen, angiotensin I, angiotensin II and renin!).
Results In sections of rat and human kidney not only the granules of the JO, but also red blood cells and some nerve fibres, were stained deep purple. In the sections from various control tissues, myelinated tracts in the brain (corpus callosum, stria medullaris), striated muscle (quadriceps, psoas and deltoid), sweat glands (Fig. 1) and the intercellular bridges of the epidermis were likewise stained. In vitro, WILSON'S crystal violet solution2 ), applied for 2 to 24 h, did not stain angiotensinogen, angiotensin I, angiotensin II or renin. 1) Rabbit serum renin-substrate (COOK et al. 1965), human serum renin-substrate (HAAS et al1966), angiotensin I (supplied by courtesy of Dr. TESSER, Department of Organic Chemistry, Uni. versity of Nijmegen), angiotensin II (hypertensin, CIBA), pig renin (PEART et al. 1966), rat renin (CUTHBERT et al. 1966). 2) 100 ml of 20 % ethyl alcohol to which has been added 10 drops of the stock solution of 1 % aqueous crystal violet. 13
Acta hi8tochcm. Bd. 58
192
G. BARBOLINI and A. C. PESSINA
Fig. 1. Human digital biopsy according to CURRI et a1. (1966): sweat glands. The secretion granules of the secretory segment (TISCHENDORF et a1. 1971) are stained deep purple by the crystal violet. X 1472.
In our opmlOn, WILSON'S crystal violet does not stain renin but probably its proteolipid carrier. Evidence that the modified crystal violet stain is able to demonstrate proteolipids is given in a separate paper.
Literature COOK, W. F., and LEE, M. R., The preparation of rabbit renin·substrate for the assay of minute amounts of renin . Biochem. J . 96,413-418 (1965). CURRI, S. B., MANZOLI, D., and TISCHENDORF, F. , Die Fingerbeerenbiopsie. Klin . Wschr. 44,584 - 590 (1966). CUTHBERT, M. F., ASSCHER, A. W., and HERY JONES, J., Characterisation of a vascular permeability factor of renal origin. Clin. Sci . 31, 325-336 (1966). FRIEDBERG, E. C., and REID, J. D., Rapid staining of the juxtaglomerular cell granules and renal tubular mitochondria for light microscopy. Nature (Lond.) 209, 1143-1144 (1966). HAAS, E., GOLDBLATT, H., GIPSON, E. E., and LA VERA LEWIS, B. S., Extraction, purification and assay of human renin free of angiotensinase. Clin. Res. 19, 739-749 (1966).
Acta
hi$to('h~m.
Plaie III
Ed. :j8
2
4
6
5
Wari ;;, H ec hardt and Parta nell VEB GUSTAV FISCHER VERLAG lENA
Acta hiatochem. Bd. 58
Plate IV
1
3a
3b
4
5
6
7
Kasabyan and Pisarev VEB GUSTAV FISCHER VERLAG lENA
Acta histochem. Ed. 58
Planche V
2
3
4
5
6
Strambi VEB GUSTAV FISCHER VERLAG JENA
Tafel VI
A ci(t hitslochem. tid. 58
1
2
3
4
5
Dunz e ndorfer und Web e r VEB GUSTAV FISCHER VERLAG lENA
Acta hi8tochern. Ed. 58
'l'afel VII
la
Passia, vVeissbach, Muller u. a.
Ib
T afel VIII
Acta histochem . Bd. 58
2a, b
l'assia. W e i ssbac h , Mull e r u . a.
Acta histochem. Bd. 58
'l'ajellX
3a-3c
Passia,
Wci~sbach,
Muller u. a.
Non-specificity of crystal violet staining
193
MARIUZZI, G_ M., and NEXCI, I., Nuova tecnica istologica per evidenziare Ie cellule granulose epitelioidi degli apparati juxtaglomerulari. Riv. Pat. Olin. Spero 7, 365-368 (1966). PEARSE, A. G. E., Histochemistry: theoretical and applied. 3rd ed. vol. 1, p. 51. J. & A. Ohurchill, London 1968. PEART, 'V. S., LLOYD, A. M., THATCHER, G. M., LEVER, A. F., PAYNE, N., and STONE, N., Purification of pig renin. Biochem. J. 99, 708-716 (1966). PITTS, R. F., Physiology of the kidney and body fluids. 2nd ed. p. 158. Year Book Med. Publ. Inc., Ohicago 1968. TISCHEXDORF, F., BARBOLINI, G., and OURRI, S. B., Histology, histochemistry and function of the human digital arteriovenous anastomoses. II. The possible relationship with sweat glands. }licrosc. Res. 3,323-336 (1971). WILSON, W., A new staining method for demonstrating the granules of the juxtaglomerular complex. Anat. Rec. 112,497-507 (1952). Address of the authors: Prof. Dr. GIUSEPPE BARBOLINI, Istituto di Anatomia e Istologia Pato10gica, Universita clegli Studi di Modena, Via Berengario, 4, I - 41100 Modena, Italia.
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