Pewarnaan mason [PDF]

tetap terwarnai, sementara unsur jaringan yang tersisa dihilangkan warnanya. Natrium tiosulfat ... Serat elastis dan int

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Pewarnaan mason Nuclei Cytoplasm, Keratin, Muscle Fibers Collagen, Mucin

Black Red Blue

Fig. 2. Longitudinal section of a cusp from a male subject showing the general layout of laminae ventricularis (V), fibrosa (F) and atrialis (A) using Mason Trichrome stain (X100). Note the arrow pointing on a strip of myocardium.

Pewarna Verhoeff juga dikenal sebagai Verhoeff’s Elastic Stain (VEG) atau Verhoeff-Van Gieson Stain (VVG) ialah suatu protokol pewarnaan yang digunakan dalam hitologi, yang dikembangkan oleh Ahli bedah optalmik dan patologis Amerika Frederick Herman Verhoeff (1874-1968) pada tahun 1908. Formulasinya digunakan untuk menunjukkan serat elastik normal atau patologis. Pewarnaan Verhoeff membentuk berbagai ikatan kation, anion dan non-ion dengan elastin, konstituen utama dari jaringan serat elastik. Elastin memiliki afinitas yang kuat untuk kompleks besi-hematoksilin yang dibentuk oleh reagen-reagen dalam pewarna itu dan karena itu akan menahan pewarna lebih lama dibandingkan unsur jaringan lain. Ini memungkinkan elastin untuk tetap terwarnai, sementara unsur jaringan yang tersisa dihilangkan warnanya. Natrium tiosulfat digunakan untuk membuang kelebihan iodium dan melawan warna (paling sering pewarna Van Gieson) digunakan untuk membedakan noda utama. Serat elastis dan inti sel berwarna hitam, serat kolagen berwarna merah dan unsur jaringan lain termasuk sitoplasma adalah berwarna kuning.



Azocarmine: Nuclei are deep red; cytoplasm is a pale red.





Azure II - Eosin (Az. II. E.): Nuclei are blue or purple. Basophilic material blue. Acidophilic material red. Red blood corpuscles orange.



Berlin Blue (Prussian Blue): An insoluble particulate iron-cyanide compound, which is used for the injection of blood and lymph vessels.



Best's Carmine: A specific stain for glycogen by which the glycogen granules are stained red. The PAS (periodic acid Schiff reaction) also colors glycogen red and is more commonly used.

 

This section of liver (note the portal tract to the lower left) was stained with Best's Carmine which stains glycogen. The bright red blotches in the cytoplasm of the hepatocytes are glycogen deposits; these are usually extracted (or stain very lightly) in most conventional H&E preparations. Compare this image with that in LIVER & GALLBLADDER 04.





Bielschowsky's Silver Method: The reticular connective tissue fibrils are black. All other structures, yellow or brown. An impregnation method, which depends on the reduction by formalin of the easily reducible silver salt, silver ammonium hydroxide.

Fig. 5a: Cross section through human spinal cord showing close-up view of internal structure of motor neuron located in the body of dorsal horn. Reduced silver method used. Silver grains are labeled 'S'. An oil immersion lens 100x has been used. (Click on image for larger version.)





Bodian Silver method: Metallic silver is precipitated by the action of a reducing agent (either exogenous or endogenous). The exogenous agent results in deposits on reticular fibers and portions of the junctional complex (argyrophilia). An endogenous agent results in precipitation on granules of enteroendocrine cells (the argentaffin reaction).  NERVE FIBERS White matter, Spinal cord cross section





Rhesus monkey, 10% formalin, Bodian silver, 612 x.







Cajal's Silver Stain (Cajal): Neurofibrils, axons and dendrites black. Other parts brown. The general principle of the Cajal methods (and there are many modification) is the application of photographic developers to tissues, which have been treated with silver nitrate.

Photomicrograph of a human spleen showing reticular tissue, section, Cajal's silver stain, mag. 36x (at 24 x 36 mm). 



Chrome Hematoxylin and Phloxin: The use of these dyes for the differential staining of the alpha and beta cells of the islets of Langerhans was described by Gomori, 1941 (Am. J. Path., Vol. 17). The granules of the beta cells are stained a deep blue; those of the alpha are pink or red. The D cells are not differentially colored.

 

This slide provides views of sections through the pancreas but with a slightly different stain, chrome-alum hematoxylin-phloxine. One should be able to appreciate all the same features detailed in the previous slide.





Cresyl violet: A basic dye. See Nissl.

 

Eosin (E): An acid dye. Colors cytoplasm red; red blood cells, orange. Used as a counterstain. See under H&E.



Foots Silver: This is a modification of Bielschowsky's silver method. The thin collagen or reticular fibers stain black, other tissues remain pale. Azocarmine is frequently used as a counter-stain to color the cells and collagenous tissue red.



Giemsa: Methylene blue eosinate, azure B eosinate, azure A eosinate, methylene blue chloride in methanol. Golgi Silver Method



(Golgi): A black deposit of reduced silver is laid down on the surfaces of nerve cells and neuroglia cells so that the form of the cell body and its processes stand out prominently in an almost colorless background. Only single cells here and there are selected by the stain. The method consists essentially of immersing fresh pieces of nervous tissue first in a solution of potassium dichromate (and usually osmic acid also) and then in silver nitrate.



Hematoxylin and Eosin (H&E): Hematoxylin is not a true basic dye. It is used with an intermediary, which recognizes anionic tissue components. Hematoxylin is nearly a specific stain for chromatin and it is therefore referred to as a "Basic" stain. It stains the nuclear network, chromosomes, etc., blue. It is a regressive stain and is extracted by very dilute acid or acid alcohol. It may be used after almost any fixative and is a permanent

stain. Eosin is a red general cytoplasmic stain. It combines with hemoglobin to give an orange color. It is an acid dye and the terms acidophilic, oxyphilic and eosinophilic are often used interchangeably. It may be used after any fixative and is used as a counter-stain in many combinations in addition to hematoxylin. 

Hematoxylin and Orange "G" (H & Or. G.): Orange G. is substituted for eosin. Acid orange-G specifically stains the granules of acidophilic cells of the adenohypophy



Nissl: A method of staining nucleic acids (e.g. ribosomes, RER, heterochromatin, nucleoli). A dye such as methylene blue, toluidine blue or cresyl violet is used. Orange "G" (Or. G.): A general cytoplasmic stain similar to eosin in action. Stains cytoplasm yellow or orange.





Slide 13: Various basic dyes demonstrate the areas of concentrated RER lamellae with free polysomes between them (together they are called Nissl bodies) as dark staining patches in the cytoplasm of neurons. Two neurons with well stained Nissl bodies are indicated by arrows. The basic dyes stain RNA particularly well and thus the nucleoli of these cells are also stained if they are in the section. There is no counterstain in this slide. (Spinal cord.)



Osmic Acid or Osmium Tetroxide (OsO4): A selective stain for unsaturated lipids and for lipoproteins such as myelin, which it stains black. Also used as a fixative, especially for electron microscopy.

 

Electron micrograph of (organic) plant tissue without (top) and with OsO4 staining



Periodic Acid Schiff and Hematoxylin (P.A.S. & H.): (See Junqueira 11th pp 12-13.) Carbohydrates and carbohydrate compounds may be demonstrated by this histochemical technique. Most carbohydrates react with periodic acid to produce aldehydes, which convert the colorless Schiff reagent to pink, or magenta. Hematoxylin or methyl green is used to stain the nuclei. Glycogen, mucin, elastic fibers, reticular fibers, basement

membranes, thyroid colloid, basophilic granules in the pituitary gland, and other polysaccharides such as the ground substance of cartilage are stained fuchsia or pink.

 

Gastric signet ring cell carcinoma histopathology, PAS stain.



Phloxin: This is a cytoplasmic stain belonging to the eosin series of dyes. It gives a reddish tone to the cytoplasm.



Mallory's phosphotungstic Acid Hematoxylin (Phostung. Hem.): Nuclei, muscle fibrils, collagenous fibers, matrix of bone and cartilage are pale red. Astrocytic glial fibrils are blue. Best after Zenker fixation.

 



Regaud's Hematoxylin for Mitochondria: Among the many methods used to demonstrate mitochondria by light microscopy, the most permanent and the simplest is Regaud's modification of iron hematoxylin on sections of material fixed in potassium dichromate and formalin and subsequently mordanted in dichromate. After staining, the slides are differentiated to remove the hematoxylin from most cytoplasmic components other than mitochondria. Unfortunately, the results are not uniform: some cells will be over-stained and some under-stained. Therefore a number of microscopic fields should be examined.

 

21. This is low power image of a section of amphibian liver stained with iron hematoxylin which stains nuclei and mitochondria a bluish-gray color. The large, brown granular cells seen here are a type of pigment cell not found in human liver so they may be disregarded.





Silver nitrate (Ag.): The intercellular cement substance of epithelium is black. This is an impregnation method. The fresh tissue is treated with silver nitrate and exposed to strong light, which reduces the silver.



Silver Method for Golgi complex: Many methods have been used for staining the Golgi complex of the cell. One of the best methods consists of direct fixation of fresh tissue in a solution of silver nitrate in formalin, development in hydroquinone-formalin, followed by the usual procedure for paraffin embedding and sectioning. In the slides prepared for the class sets, the nuclei of the cells have been stained lightly by azocarmine.



Sudan Black: This is a stain that colors fat droplets black. There are several Sudan dyes, among which are Sudan III and Sudan IV (Scarlet R.). These stain fat droplets red as does Oil-red-O.



Supravital staining: A vital stain (e.g., trypan blue) is applied to an animal in life; a supravital stain (e.g., Janus green, neutral red) is one that is applied to cells or tissues removed from the body. See the first laboratory exercise, Introduction: Microscopy -

Cytology for a description of the properties of neutral red and Janus green vis a vis particular subcellular organelles. 



Supravital stain of a smear of human blood from a patient with hemolytic anemia. The reticulocytes are the cells with the dark blue dots and curved linear structures (reticulum) in the cytoplasm.



Toluidine blue: A basic dye. See Nissl.

 

Van Gieson: Collagenous fibers are yellow (e.g., dense connective tissue and bone), cartilage matrix is brown. Elastic Tissue Stain and Van Gieson's Picric Acid Fuchsin (Vieh. Van G. Verhoeff's): In Verhoeff's stain the elastic tissue is black and in this combination with Van Gieson collagenous tissue is red.

 

Figure 4: Verhoeff-van Gieson stain for elastin showing multiple small areas of diminished and fragmented elastic tissue. (Verhoeff-van Gieson, ×40)



Weigert's Hematoxylin (W. Hem.): A modification of Heidenhain's iron hematoxylin. Stains the myelin sheath black.



Weigert's Elastic Tissue Stain: Elastic fibers purple or black. It is a specially prepared combination of basic fuchsin, resorcin, ferric chloride, water and alcohol.





Fig. 4. Longitudinal sections of cusps from males with (a) showing decreased myocardial fibres in the middle zone (Weigert's elastic stain, X400) and (b) showing elastic fibre lamellae and absence of muscle at the edge (Weigert's elastic stain, X400). 



Wright's Blood Stain (Wright): Nuclei blue or purple. Basophilic granules blue, acidophilic granules red, and neutrophilic granules reddish lilac. Red blood corpuscles orange. The eosinates of polychromed methylene blue are dissolved in absolute methyl alcohol. When this solution is placed on a dried blood smear, the methyl alcohol acts as the fixative, and the dissolved dye begins the staining process. After from one to three minutes, the stain is diluted with an equal volume of distilled water. This differentially stains the cytoplasmic granules and is allowed to act for about three minutes. It is then poured off and the preparation is washed briefly in tap water and allowed to dry

 

Micrograph of smear of monkey blood; Wright's stain (Lab slide L)



Azocarmine: Nuclei yang merah tua; sitoplasma adalah merah pucat. Azure II - Eosin (Az II E...): Nuclei berwarna biru atau ungu. Basophilic bahan biru. Bahan acidophilic merah. Sel-sel darah merah oranye. Berlin Biru (Prussian Blue): Sebuah larut senyawa partikulat besi-sianida, yang digunakan untuk injeksi pembuluh darah dan getah bening. Best Carmine: Noda khusus untuk glikogen dimana butiran glikogen berwarna merah. PAS (periodik reaksi Schiff asam) juga warna glikogen merah dan lebih sering digunakan. Metode Bielschowsky Perak: The reticular fibril jaringan ikat berwarna hitam. Semua struktur lainnya, kuning atau coklat. Sebuah metode impregnasi, yang tergantung pada pengurangan oleh formalin dari mudah direduksi garam perak, perak amonium hidroksida. Metode Perak Bodian: Metallic perak diendapkan oleh aksi zat pereduksi (baik eksogen atau endogen). Hasil agen eksogen dalam deposito pada serat retikuler dan bagian-bagian dari junctional kompleks (argyrophilia). Hasil agen endogen curah hujan pada butiran sel enteroendokrin (reaksi argentaffin). Cajal Perak Stain (Cajal): Neurofibrils, akson dan dendrit hitam. Bagian lain coklat. Prinsip umum metode Cajal (dan ada banyak modifikasi) adalah aplikasi pengembang fotografi untuk jaringan, yang telah diperlakukan dengan perak nitrat. Chrome Hematoxylin dan Phloxin: Penggunaan pewarna ini untuk pewarnaan diferensial sel alfa dan beta dari pulau Langerhans digambarkan oleh Gomori 1941 (Am J. jalan, Vol 17...). Butiran sel beta berwarna biru tua; orang-orang dari alpha yang merah muda atau merah. Sel-sel D tidak berwarna berbeda-beda. Cresyl violet: Sebuah pewarna dasar. Lihat Nissl. Eosin (E): Sebuah pewarna asam. Warna sitoplasma merah; sel darah merah, oranye. Digunakan sebagai counter-noda. Lihat di bawah H & E. Endapan Silver: ini merupakan modifikasi dari metode Bielschowsky perak. Kolagen tipis atau reticular serat noda hitam, jaringan lain tetap pucat. Azocarmine sering digunakan sebagai counter-noda untuk mewarnai sel dan jaringan kolagen merah. Giemsa: Methylene eosinate biru, biru B eosinate, biru A eosinate, klorida biru metilen dalam metanol. Metode Golgi Perak (Golgi): Deposit hitam berkurang perak diletakkan di atas permukaan sel-sel saraf dan sel neuroglia sehingga bentuk sel tubuh dan proses yang menonjol jelas di latar belakang

hampir tidak berwarna. Hanya sel tunggal di sana-sini yang dipilih oleh noda. Metode ini pada dasarnya terdiri dari merendam potongan segar dari jaringan saraf pertama dalam larutan kalium dikromat (dan asam biasanya osmik juga) dan kemudian di perak nitrat. Hematoxylin dan Eosin (H & E): Hematoksilin bukan pewarna dasar yang benar. Hal ini digunakan dengan perantara, yang mengakui komponen jaringan anionik. Hematoxylin hampir noda khusus untuk kromatin dan oleh karena itu disebut sebagai "Basic" noda. Ini noda jaringan nuklir, kromosom, dll, biru. Ini adalah noda regresif dan diekstraksi dengan asam encer atau alkohol sangat asam. Ini dapat digunakan setelah hampir semua fiksatif dan noda permanen. Eosin adalah noda sitoplasma umum merah. Ini menggabungkan dengan hemoglobin untuk memberikan warna oranye. Ini adalah zat warna asam dan persyaratan acidophilic, oxyphilic dan eosinophilic sering digunakan secara bergantian. Ini dapat digunakan setelah fiksatif apapun dan digunakan sebagai counter-noda dalam banyak kombinasi selain hematoxylin. Hematoxylin dan Orange "G" (H & Atau G..): Jeruk G. diganti untuk eosin. Asam jeruk-G khusus noda butiran sel acidophilic dari adenohypophy yang 



Nissl: Sebuah metode pewarnaan asam nukleat (misalnya ribosom, RER, heterochromatin, nukleolus). Sebuah pewarna seperti methylene blue, toluidin biru atau ungu cresyl digunakan. Oranye "G" (Atau G..): Noda sitoplasma umum mirip dengan eosin dalam tindakan. Noda sitoplasma kuning atau oranye. Asam osmik atau Osmium ferri (OsO4): Noda selektif untuk lipid tak jenuh dan lipoprotein seperti myelin, yang noda hitam. Juga digunakan sebagai fiksatif, terutama untuk mikroskop elektron. Periodic Acid Schiff dan Hematoksilin (PAS & H.): (Lihat Junqueira 11 pp 12-13.) Karbohidrat dan senyawa karbohidrat dapat ditunjukkan dengan teknik histokimia ini. Kebanyakan karbohidrat bereaksi dengan asam periodik untuk menghasilkan aldehida, yang mengkonversi berwarna Schiff reagen untuk merah muda, atau magenta. Hematoxylin atau metil hijau digunakan untuk noda inti. Glikogen, mucin, serat elastis, serat reticular, membran basement, koloid tiroid, butiran basophilic di kelenjar hipofisis, dan polisakarida lain seperti substansi dasar tulang rawan yang fuchsia bernoda atau merah muda. Phloxin: Ini adalah noda sitoplasma milik seri eosin pewarna. Ini memberikan nada kemerahan ke sitoplasma. Mallory fosfotungstat Asam Hematoksilin (Phostung Hem..): Nuclei, fibril otot, serat kolagen, matriks tulang dan tulang rawan merah pucat. Fibril glial astrositik berwarna biru. Terbaik setelah Zenker fiksasi. Hematoxylin Regaud untuk Mitokondria: Di antara banyak metode yang digunakan untuk menunjukkan mitokondria dengan mikroskop cahaya, yang paling permanen dan

sederhana adalah modifikasi Regaud dari besi hematoxylin pada bagian bahan tetap kalium dikromat dan formalin dan kemudian mordanted di dikromat. Setelah pewarnaan, slide dibedakan untuk menghapus hematoxylin dari sebagian komponen sitoplasma selain mitokondria. Sayangnya, hasilnya tidak seragam: beberapa sel akan lebih bernoda dan beberapa di bawah bernoda. Oleh karena itu sejumlah bidang mikroskopis harus diperiksa. Perak nitrat (Ag.): Substansi semen antar epitel hitam. Ini adalah metode impregnasi. Jaringan segar diperlakukan dengan perak nitrat dan terkena cahaya yang kuat, yang mengurangi perak. Metode perak untuk Golgi kompleks: Banyak metode telah digunakan untuk pewarnaan kompleks Golgi sel. Salah satu metode terbaik terdiri dari fiksasi langsung jaringan segar dalam larutan perak nitrat dalam formalin, pembangunan di hydroquinoneformalin, diikuti oleh prosedur biasa untuk embedding parafin dan sectioning. Dalam slide disiapkan untuk set kelas, inti sel telah ternoda ringan oleh azocarmine. Sudan Black: Ini adalah noda yang warna tetesan lemak hitam. Ada beberapa pewarna Sudan, di antaranya adalah Sudan III dan IV Sudan (Scarlet R.). Ini noda lemak tetesan merah seperti halnya minyak-red-O. Supravital pewarnaan: Noda penting (misalnya, tripan biru) diterapkan untuk binatang dalam kehidupan; noda supravital (misalnya, Janus hijau, merah netral) adalah salah satu yang diterapkan pada sel-sel atau jaringan dikeluarkan dari tubuh. Lihat latihan laboratorium pertama, Pendahuluan: Mikroskop - Sitologi untuk penjelasan dari sifatsifat netral merah dan Janus hijau vis a vis organel subselular tertentu. Toluidin biru: Sebuah pewarna dasar. Lihat Nissl. Van Gieson: serat Collagenous kuning (misalnya, jaringan ikat padat dan tulang), tulang rawan matriks berwarna coklat. Elastis Tissue Stain dan Van Gieson ini Picric Acid Fuchsin (Vieh Van G. Verhoeff ini.): Dalam noda Verhoeff dunia jaringan elastis hitam dan dalam kombinasi dengan Van Gieson jaringan kolagen merah. Weigert ini Hematoxylin (W. Hem.): Sebuah modifikasi dari hematoxylin besi Heidenhain ini. Noda selubung mielin hitam. Weigert Elastic Tissue Stain: serat elastis ungu atau hitam. Ini adalah kombinasi khusus disiapkan dari fuchsin dasar, resorcin, besi klorida, air dan alkohol. Wright Blood Stain (Wright): Nuclei biru atau ungu. Basofilik granul biru, butiran acidophilic merah, dan butiran neutrophilic kemerahan ungu. Sel-sel darah merah oranye. The eosinates dari polychromed biru metilen dilarutkan dalam metil alkohol absolut. Ketika solusi ini ditempatkan pada smear darah kering, alkohol metil bertindak sebagai fiksatif, dan pewarna terlarut dimulai proses pewarnaan. Setelah dari satu sampai tiga menit, noda diencerkan dengan volume yang sama dari air suling. Ini berbeda-beda noda

butiran sitoplasma dan diperbolehkan untuk bertindak selama sekitar tiga menit. Hal ini kemudian dituangkan off dan persiapan dicuci sebentar dalam air keran dan dibiarkan kering

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