Introduction
- Connective aur dusre mesenchymal tissues body ke chaar basic tissue types me se ek hai, jo epithelial, muscular, aur nervous tissues ke saath aata hai.
- Yeh primarily body ke structure aur function ko support dene, tissues aur organs ko connect aur bind karne ka kaam karta hai.
- Connective tissue cells aur extracellular matrix (ECM) se bana hota hai, jisme fibers (collagen, elastic, reticulin) aur ground substance hota hai.
- Mesenchymal tissues connective tissues ka ek subset hote hain, jo mesodermal progenitor cells se embryogenesis ke dauran develop hote hain.
- Yeh tissues bones, cartilage, tendons, aur muscles jaise body structures ke formation mein important hote hain.
- Mesenchymal tissues tissue repair aur regeneration mein bhi important role play karte hain poori life ke dauran.
Features of Connective aur Mesenchymal Tissues
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Cells: Connective aur mesenchymal tissues me fibroblasts, chondrocytes, osteocytes, adipocytes, aur macrophages cells hote hain.
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Extracellular Matrix (ECM): ECM fibers (collagen, elastic, reticulin) aur ground substance se bana hota hai. ECM ki composition tissue ki mechanical properties determine karti hai, jaise bones me rigidity ya ligaments me elasticity.
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Fibers:
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Collagen fibers: Yeh tensile strength aur structural integrity provide karte hain.
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Elastic fibers: Yeh elasticity provide karte hain, jo tissues ko stretch aur apni original shape me wapas aane ka chance dete hain.
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Reticulin fibers: Yeh delicate mesh banate hain, jo organs jaise liver, spleen, aur lymph nodes ko support karte hain.
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Types of Connective Tissues
Connective tissues ko broadly do categories mein classify kiya ja sakta hai:
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Loose Connective Tissue: Isme areolar, adipose, aur reticular tissue aate hain. Yeh tissues cushioning aur support provide karte hain.
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Dense Connective Tissue: Isme zyada collagen fibers hote hain aur yeh strength aur flexibility provide karte hain. Yeh tendons aur ligaments mein paaye jaate hain.
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Specialized Connective Tissues: Jaise cartilage, bone, aur blood, jo specific functions perform karte hain.
Staining Methods
Periodic Acid-Methenamine Silver Microwave Method
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Fixative: 10% neutral buffered formalin preferred hai. Mercury-containing fixatives nahi recommend kiye jaate.
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Sections: Paraffin-processed tissue ko 2 μm tak cut kiya jaata hai.
Solutions:
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Stock Methenamine Silver:
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3% aqueous methenamine – 400 ml
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5% aqueous silver nitrate – 20 ml
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Refrigerate at 4°C.
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5% Borax (Sodium Borate) Solution
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Working Methenamine Silver Solution:
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Stock methenamine silver – 25 ml
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Distilled water – 25 ml
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5% borax – 2 ml
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1% Periodic Acid Solution
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0.2% Gold Chloride Solution:
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1% gold chloride – 1 ml
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Distilled water – 49 ml
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Stock Light Green Solution:
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Light green SF (yellowish) – 1 g
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Distilled water – 500 ml
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Glacial acetic acid – 1 ml
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Working Light Green Solution:
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Light green stock solution – 10 ml
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Distilled water – 50 ml
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Method:
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Sections ko deparaffinize karke distilled water mein rehydrate karen.
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1% periodic acid solution mein 15 minutes tak rakhen.
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Distilled water mein rinse karen.
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50 ml methenamine working solution mein 5 slides ko rakhkar microwave karein. 70 seconds ke liye microwave karen.
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Desired staining intensity achieve hone tak 15-20 minutes lagte hain.
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Rinse slides in heated distilled water.
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Tone sections ko 0.02% gold chloride mein 30 seconds ke liye.
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Rinse in distilled water.
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2% sodium thiosulfate se 1 minute tak treat karen.
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Tap water se wash karein.
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Counterstain working light green solution mein 1.5 minutes tak karein.
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Dehydrate with two changes of 95% aur absolute alcohol.
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Xylene se clear karen aur synthetic resin se mount karen.
Results:
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Basement membrane: Black
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Background: Green
Van Gieson Technique
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Sections: Paraffin. Double embedding celloidin ya low-viscosity nitrocellulose (LVN) sections mein hota hai.
Van Gieson Solution:
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Saturated aqueous picric acid solution – 50 ml
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1% aqueous acid fuchsin solution – 9 ml
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Distilled water – 50 ml
Method:
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Sections ko deparaffinize karen aur water mein le aayein.
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Nuclei ko celestine blue-hematoxylin sequence se stain karein.
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Tap water mein wash karein.
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Acid alcohol mein differentiate karen.
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Achhe se tap water mein wash karein.
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Van Gieson solution mein 3 minutes tak stain karein.
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Dehydrate karen through ascending grades of alcohol.
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Xylene se clear karen aur permanent mounting medium mein mount karein.
Results:
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Nuclei: Blue/black
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Collagen: Red
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Other tissues: Yellow
Masson Trichrome Technique
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Fixation: Formal sublimate ya formal saline.
Preparation of Solutions:
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Solution A:
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Acid fuchsin – 0.5 g
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Glacial acetic acid – 0.5 ml
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Distilled water – 100 ml
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Solution B:
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Phosphomolybdic acid – 1 g
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Distilled water – 100 ml
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Solution C:
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Methyl blue – 2 g
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Glacial acetic acid – 2.5 ml
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Distilled water – 100 ml
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Method:
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Sections ko deparaffinize karen aur water mein le aayein.
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Mercury pigment ko iodine aur sodium thiosulfate sequence se remove karen.
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Tap water mein wash karein.
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Nuclei ko celestine blue-hematoxylin method se stain karein.
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1% acid alcohol se differentiate karein.
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Wash well in tap water.
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Acid fuchsin solution mein 5 minutes ke liye stain karein.
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Rinse in distilled water.
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Phosphomolybdic acid solution B mein 5 minutes ke liye treat karen.
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Drain karein.
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Methyl blue solution C mein 2–5 minutes tak stain karen.
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Rinse in distilled water.
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1% acetic acid mein 2 minutes tak treat karen.
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Dehydrate through ascending grades of alcohol.
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Xylene se clear karen aur permanent mounting medium mein mount karen.
Results:
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Nuclei: Blue/black
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Cytoplasm, muscle, aur erythrocytes: Red
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Collagen: Blue
Verhöeff’s Method for Elastic Fibers
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Preparation of Stain:
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Solution A: Hematoxylin – 5 g, Absolute alcohol – 100 ml
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Solution B: Ferric chloride – 10 g, Distilled water – 100 ml
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Solution C, Lugol’s iodine: Iodine – 1 g, Potassium iodide – 2 g, Distilled water – 100 ml
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Verhöeff’s Solution: Mix Solution A (20 ml), Solution B (8 ml), Solution C (8 ml).
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Method:
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Sections ko deparaffinize karen aur water mein le aayein.
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Verhöeff’s solution mein 15–30 minutes ke liye stain karen.
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Water mein rinse karein.
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2% aqueous ferric chloride mein differentiate karen jab tak elastic fibers black na ho jayein.
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Water mein rinse karein.
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Xylene se clear karen aur permanent mounting medium mein mount karen.
Results:
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Elastic tissue fibers: Black
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Other tissues (counterstain ke according)
Aldehyde Fuchsin Method for Elastic Fibers
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Preparation of Staining Solution:
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1 g basic fuchsin ko 100 ml 70% ethanol mein dissolve karen.
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1 ml concentrated HCl aur 2 ml paraldehyde add karen.
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2 din ke liye room temperature par rakhen.
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Solution ka color change hoga red se purple mein.
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Method:
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Sections ko deparaffinize karen aur water mein le aayein.
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1% potassium permanganate mein 5 minutes ke liye oxidize karen.
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Tap water mein rinse karen.
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1% oxalic acid se permanganate staining remove karen.
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Tap water mein rinse karen.
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70% ethanol mein rinse karen.
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Sealed container mein aldehyde fuchsin mein 15 minutes tak rakhkar stain karein.
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70% ethanol mein rinse karen.
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Tap water mein rinse karen.
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Counterstain eosin, van Gieson ya neutral red ke saath kar sakte hain.
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Alcohol se dehydrate karein.
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Xylene se clear karen aur permanent mounting solution mein mount karen.
Results:
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Elastic tissue fibers: Blue-purple
Gordon & Sweets’ Method for Reticular Fibers
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Preparation of Silver Solution:
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5 ml 10% aqueous silver nitrate mein concentrated ammonia drop by drop add karein.
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Add 5 ml 3% sodium hydroxide solution.
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Solution ko 50 ml distilled water se volume tak le aayein.
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Filter karen aur dark plastic bottle mein store karein.
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Method:
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Sections ko deparaffinize karen aur water mein le aayein.
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1% potassium permanganate solution mein 5 minutes ke liye treat karen.
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Tap water mein rinse karein.
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1% oxalic acid solution se bleach karein.
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Tap water mein rinse karein.
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2.5% iron alum solution mein 15 minutes tak treat karen.
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Distilled water se wash karen.
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Silver solution mein 2 minutes ke liye treat karen.
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Distilled water se rinse karein.
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10% aqueous formalin solution mein 2 minutes tak reduce karen.
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Tap water mein rinse karein.
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0.2% gold chloride solution mein 3 minutes ke liye tone karen.
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Tap water mein rinse karein.
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5% sodium thiosulfate solution se 3 minutes ke liye treat karen.
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Tap water mein rinse karein.
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Counterstain as desired.
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Alcohol se dehydrate karein.
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Xylene se clear karen aur permanent mounting medium mein mount karen.
Results:
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Reticular fibers: Black
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Nuclei: Black ya unstained
Gomori’s Method for Reticular Fibers
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Preparation of Silver Solution:
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40 ml 10% silver nitrate solution mein 10 ml 10% potassium hydroxide solution add karen.
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Precipitate settle karne de aur supernatant decant karein.
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Ammonia drop by drop add karein until precipitate dissolve ho jaye.
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10% silver nitrate solution aur distilled water se dilute karen.
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Method:
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Sections ko deparaffinize karen aur water mein le aayein.
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1% potassium permanganate solution mein 2 minutes ke liye treat karen.
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Tap water mein rinse karen.
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2% potassium metabisulfate solution se bleach karen.
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Tap water mein rinse karen.
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2% iron alum solution mein 2 minutes tak treat karen.
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Distilled water se wash karen.
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Silver solution mein 1 minute ke liye treat karen.
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Distilled water se rinse karein.
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4% aqueous formalin solution mein 3 minutes ke liye reduce karen.
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Tap water mein rinse karein.
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0.2% gold chloride solution mein 10 minutes tak tone karen.
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Tap water mein rinse karein.
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2% potassium metabisulfate solution mein 1 minute ke liye treat karen.
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Tap water mein rinse karein.
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2% sodium thiosulfate solution mein 1 minute ke liye treat karen.
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Tap water mein rinse karein.
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Counterstain as desired.
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Alcohol se dehydrate karen.
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Xylene se clear karen aur permanent mounting medium mein mount karen.
Results:
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Reticular fibers: Black
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Nuclei: Gray