microtome A microtome is used for cutting thin sections. It is just a with a mechanism that allow the sharp knife advancing of a paraffin block standard distances across it. Cut sections are floated on water in a water bath at 50 C, then picked on glass slides. Sections come as a ribbon paraffin sections picked on slide. The glass slides are then placed in a warm oven for about 15 minutes to help the section adhere to the slide Staining of sections: H & E is commonly used to stain paraffin sections: is a basic stain. It stains basophilic structures such as chromatin and ribosome's Hematoxylin a deep or purple blue. The advantage of this stain is that it provides a clear stain of the cell nuclei. is an that stains It provides a good stain for the Eosin acidic stain acidophilic structures red. cell membrane Reagents: -Hematoxylin (Sigma Accustain Harris Hematoxylin) – -Acidic Alcohol (396 ml of 95% Ethanol, 4 mls concentrated HCl) -Bluing Agent (1 liter water, 1 gm sodium bicarbonate) -Eosin - Sigma Accustain Eosin Y -75%, 95%, 100% ethanol -Histo-clear (order No. HS 200) -Permount Staining procedure: Hematoxylin 5 minutes – o Rinse in tap water 30 seconds, to remove excess stain. – o Acidic Alcohol 5 dips, to and destain differentiate. – o Rinse in tap water 30 seconds – o Bluing Agent 2 minutes. This step is optional. The sodium bicarbonate changes the color of the – hematoxylin-stained nuclei from purple to blue.
microtome A microtome is used for cutting thin sections. It is just a sharp knife with a mechanism that allow the advancing of a paraffin block standard distances across it. Cut sections are floated on water in a water bath at 50 C, then picked on glass slides. Sections come as a ribbon paraffin sections picked on slide. The glass slides are then placed in a warm oven for about 15 minutes to help the section adhere to the slide Staining of sections: H & E is commonly used to stain paraffin sections: Hematoxylin is a basic stain. It stains basophilic structures such as chromatin and ribosome's a deep purple or blue. The advantage of this stain is that it provides a clear stain of the cell nuclei. Eosin is an acidic stain that stains acidophilic structures red. It provides a good stain for the cell membrane Reagents: -Hematoxylin – (Sigma Accustain Harris Hematoxylin) -Acidic Alcohol (396 ml of 95% Ethanol, 4 mls concentrated HCl) -Bluing Agent (1 liter water, 1 gm sodium bicarbonate) -Eosin - Sigma Accustain Eosin Y -75%, 95%, 100% ethanol -Histo-clear (order No. HS 200) -Permount Staining procedure: Hematoxylin – 5 minutes o Rinse in tap water – 30 seconds, to remove excess stain. o Acidic Alcohol – 5 dips, to destain and differentiate. o Rinse in tap water – 30 seconds o Bluing Agent – 2 minutes. This step is optional. The sodium bicarbonate changes the color of the hematoxylin-stained nuclei from purple to blue.
Cell%20Biology%20Lab%20Manual.docx