How can we identify the organoids we have painstakingly cultured? Typically, this involves HE staining and immunohistochemical staining. Today, the editor will take you through the process of how to embed organoids and perform HE staining.

 

I. Organoid Collection

 

1. Organoid Collection (e.g., collect 1-2 wells of organoids from a 24-well plate, approximately 200-500 organoids);

2. Stand at 4°C for 10 minutes, centrifuge and discard the majority of the supernatant.

 

II. Preparation of Agarose Wells

1. Weigh 0.2 to 0.4g of agarose and add it to a beaker or a small reagent bottle;

2. Add approximately 10mL of PBS;

3. Melt in a microwave at high power, pausing every 5 seconds to check, repeat this process several times until the agarose is completely melted;

4. Take about 200μL of melted agarose and add it to a 1.5mL EP tube (as shown in the figure below);

5. Insert a small 0.5mL EP tube into the 1.5mL EP tube containing the agarose to form a round-bottomed well, let it stand at room temperature or in a 4°C refrigerator for a few minutes until the agarose solidifies. Gently rotate the small 0.5mL EP tube and remove it, revealing a deep round-bottomed well in the agarose;

6. Pipette the organoid pellet to the bottom of the well, trying to embed it in the center;

7. Add PBS until it is higher than the edge of the agarose well.

III. Organoid Fixation

IV. Embedding of Organoids

1. Dehydration: Remove the solidified agarose block containing the organoid pellet and dehydrate it in a graded series of ethanol solutions, 1 hour each in 70%, 80%, 90%, and 95% ethanol, and 30 minutes in 100% ethanol, allowing it to solidify;

2. Wax melting: Turn on the oven about 4 hours in advance and melt the wax at 65°C;

3. Clarification: After dehydration, transfer the agarose block to a tissue embedding cassette (keep it immersed in anhydrous ethanol until it is treated with xylene), transfer the cassette to xylene for 5 minutes for two treatments; Note: Xylene is toxic, so perform the operation in a well-ventilated area and try to drain any residual xylene when removing it;

4. Wax infiltration: Immediately after clarification, transfer the block to the melted wax in a wax pot, and wax infiltrate at 60°C for 2 hours, then replace with fresh wax and infiltrate for another 2 hours;

5. Embedding: Perform the embedding on an ice box, pour the melted pure wax into the cassette to one-third of its height, place the agarose block in the center, and then continue to pour wax until the cassette is filled. Once the wax block is completely solidified, carefully remove the mold.

 

V. HE Staining of Organoids

 

1. Deparaffinization of wax sections to water

Place the sections sequentially in the following solutions: Xylene I for 10 minutes; Xylene II for 10 minutes; Anhydrous ethanol I for 5 minutes; Anhydrous ethanol II for 5 minutes; 95% alcohol for 5 minutes; 90% alcohol for 5 minutes; 80% alcohol for 5 minutes; 70% alcohol for 5 minutes; Rinse with distilled water.

 

2. Hematoxylin staining of cell nuclei

Submerge the sections in Harris hematoxylin stain for 3-8 minutes, then rinse with tap water.

 

3. Eosin staining of cytoplasm

Submerge the sections in eosin stain for 1-3 minutes, then rinse with tap water.

 

4. Dehydration and mounting

Place the sections sequentially in the following solutions for dehydration and clarification: 95% alcohol I for 5 minutes; 95% alcohol II for 5 minutes; Anhydrous ethanol I for 5 minutes; Anhydrous ethanol II for 5 minutes; Xylene I for 5 minutes; Xylene II for 5 minutes. Remove the sections from xylene and let them air dry slightly before mounting with neutral balsam.

 

5. Microscopic examination, image acquisition, and analysis

 

VI. Organoid Identification - HE Staining Results (Lung Cancer as an Example)

Human Lung Cancer Organoid HE Staining

 

Item NO.	Product Name	Size
abs9514	Mouse Intestinal Organoid Culture Medium Kit	1kit
abs9516	Mouse Liver Organoid Culture Medium Kit	1kit
abs9515	Mouse Lung Organoid Culture Medium Kit	1kit
abs9446	Human Breast Cancer Organoid Culture Medium Kit	1kit
abs9448	Human Endometrial carcinoma Organoid Culture Medium Kit	1kit
abs9445	Human Colorectal cancer Organoid Culture Medium Kit	1kit
abs9443	Human Lung Cancer Organoid Culture Medium Kit	1kit
abs9449	Human Gastric Cancer Organoid Culture Medium Kit	1kit
abs9447	Human Pancreatic Cancer Organoid Culture Medium Kit	1kit
abs9558	Human Ovarian Cancer Organoid Culture Medium Kit	1kit
abs9544	Human Liver Organoid Culture Medium Kit	1kit
abs9546	Human Lung Organoid Culture Medium Kit	1kit
abs9549	Mouse Gastric Cancer Organoid Culture Medium Kit	1kit
abs9550	Mouse Lung Cancer Organoid Culture Medium Kit	1kit
abs9551	Mouse Breast Cancer Organoid Culture Medium Kit	1kit
abs9552	Mouse Hepatocarcinoma Organoid Culture Medium Kit	1kit
abs9553	Mouse Pancreatic Cancer Organoid Culture Medium Kit	1kit

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.cn

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