Glutaraldehyde fixative (2.5%, for electron microscopy)
The purpose of fixation is to preserve the original morphological structure of cells and tissues.fixercan prevent autolysis of endogenous lysosomal enzymes on their own tissues and cells and inhibit bacterial and fungal growth. Fixatives alter the internal structure of proteins through coagulation, formation of added compounds, and the like. This inactivates the enzyme. The fixative physically alters the extracellular components of the cells.
Fixatives are mainly divided into aldehyde fixative, mercury fixative, alcohol fixative, oxidant fixative and picrate fixative. Formalin in aldehydes and ethanol in alcohols are more commonly used. Glutaraldehyde fixative can cause deformation of the protein α-helix structure, which is not conducive to peroxidase staining.GlutaraldehydFixing agent has fast fixing speed and poor permeability.
How to use:
1. Work according to the specific needs of the experiment.
2. Collect fresh specimens and immediately fix them in glutaraldehyde fixative at 4°C for 1 to 4 hours. For slightly larger samples, the fixation time should be increased accordingly.
3. Submit for inspection or store at 4℃.
1. Glutaraldehyde fixative is corrosive to a certain extent. Please work carefully in a well-ventilated environment to avoid inhalation.
2. The thickness of the fabric is different, and the fixing time is also different. The appropriate thickness of conventional biopsy tissue is 2 to 4 mm, generally no more than 6 mm. Proper selection of tissues is beneficial for fixative penetration.
3. The volume of the fixative should be sufficient. In general, the volume ratio of fixative to tissue block should be greater than 10:1. If the volume is not sufficient, the fixing solution can be changed 1 to 3 times during the fixing time.
4. The effect of temperature on fixation is obvious. Raising the temperature can speed up the fixation, but it is better not to raise the temperature of this fixative.
5. After the fresh tissue is taken, it should be fixed in time. If it cannot be fixed in time, it should be kept in physiological saline and sent for examination in time.
6. For your safety and health, please wear lab coat and disposable gloves for operation.
To study the effect of different fixatives on the effect of scanning electron microscopy on biological samples.
A scanning electron microscope has the characteristics of high resolution, large depth of field and wide range of imaging magnification. Therefore, it is more and more widely used in biology, medicine, chemical industry, geology and materials.
In order to better observe the microstructure of the material, the sample must first be treated before observation. The effect of different fixatives on the processing of the sample is important.
To study the effect of different fixatives on the effect of scanning electron microscopy on biological samples. Four fixatives were made by the researchers. They were 10% formalin, Carnot's solution, 2.5% glutaraldehyde, 2.5% glutaraldehyde, and 1% starvation acid double fixative.
Root tips of the same thickness and length of 8mm were taken. After cleaning, they were fixed in each of the four fixatives mentioned above. According to the material fixing time requirements of different fixing agents.
When the final fixing time is reached, dehydration, drying and gold spraying are carried out. The better site of the root tip was selected and observed, measured and photographed for comparison with the same magnification.
Summary of test results.
1. Effect of different fixatives on biological samples
It has been widely used as a good simple fixative in paraffin sections. It is commonly used in the collection and preservation of plant and animal materials. The properties of formalin on tissue materials are strong penetration and less tissue shrinkage. It can also form adducts with proteins and has a preservative effect on lipids.
Formalin fixative is more suitable for fixing mitochondria and Golgi complexes in cells. It is also a preservative for sugar. Formalin is a reducing agent. It can be mixed with water, alcohol and ether in any proportion.
1.2 Carnot liquid.
Carnot liquid is made from pure alcohol, glacial acetic acid, and chloroform. And in this experiment, the liquid Carnot fixative was made using only alcohol and glacial acetic acid (4:1).
Alcohol is a reducing agent, a good preservative, and a common dehydrating agent, and can be mixed with water in any proportion. Alcohol can precipitate proteins, nucleic acids and liver sugar. It can dissolve fats and lipids, damage mitochondria and Golgi complexes, and reduce the size of tissues when contracted more vigorously.
Sclerosis of tissues fixed by alcohol is significant. Biological tissue left in high concentrations of alcohol for too long can become brittle.
Glacial acetic acid precipitates nucleoproteins and mucus and destroys mitochondria in the cytoplasm. It causes a slight swelling of the tissues. Thus, the shrinkage formed by other reagents can be washed out in the mixed fixative.
Glutaraldehyde fixative penetrates tissues quickly and can better preserve proteins and glycogen. It is good at preserving structures such as microtubules and smooth endoplasmic reticulum. However, glutaraldehyde fixative has no “electron staining” effect on tissue. If the material is fixed and treated with conductivity, it can achieve good results.
1.4 Starvation acid is a strong oxidizing agent. It can fix protein molecules, but it can also fix fat and lipoproteins. It has a greater affinity for cell membranes, mitochondria, etc. and has an "electron staining" effect.
2. Differences in the appearance of the same material in different fixatives
It can be seen from the images taken at the same magnification: the clarity of the images and the thickness of the material are essentially the same for the material fixed with formalin or glutaraldehyde. The shrinkage of the material fixed with Carnot's solution was more pronounced. While the material fixed with glutaraldehyde and hunger acid tended to be normal with clear cell borders. This suggests that different fixatives produced different effects on plant root tips.
It is well known that plant root tip cells divide vigorously and metabolize rapidly. At this time, they are susceptible to changes in the presence of external stimuli. This is related to the fixative used.
For example, Carnot's fixative, which has a high specific gravity of alcohol, destroys the lipids and organelles in the cytoplasm. Glacial acetic acid is supposed to swell the material. However, the difference in the ratio between the two is large. Eventually, this leads to changes in inclusions in the root tip. Thus, it shows a significant change in the image under the same conditions.
How to choose a fixative when preparing SEM samples
Retention of the original shape of the material is a basic requirement for the preparation of samples of various biological materials (from small unicellular blood cells, bacteria and algae to large plant and animal tissues). The choice of fixative is one of the keys to sample preparation. Different units can choose the required fixative according to the specific situation and the final needs of the sample.
If money is sufficient and sample accuracy is high, it is appropriate to use glutaraldehyde and starvation acid double fixation. If money is lacking and material requirements are not so delicate, formalin or glutaraldehyde can be used as a fixative. A satisfactory observation effect can also be obtained. Since the material changes greatly when the material is treated with Carnot's solution, it is generally not suitable.
Glutaraldehyde fixative for electron microscopy
9. September 2022