New fixation method for brown fat samples for transmission electron microscopy

Abstract

The present invention proposes a new fixation method for transmission electron microscopy samples of brown adipose tissue to obtain better ultrastructures of mitochondria and lipid droplets. The fixation method of brown adipose tissue of rodents is optimized, mainly including increasing the concentration of paraformaldehyde in the pre-fixative from 2% to 4%; replacing the buffer of the post-fixative osmium tetroxide with an imidazole buffer; and adopting a fixation method combining cardiac perfusion and immersion. The structures of mitochondrial cristae and other membranous organelles of brown fat cells prepared by the new fixation method are more complete; lipid droplets appear as round structures with high electron density. The method was used to observe the increase in the density of mitochondrial cristae and the increase in lipid droplets in brown fat cells after cold adaptation. The new fixation method can better preserve the ultrastructure of organelles in brown fat cells, especially mitochondria and lipid droplets, and can be used to study the ultrastructural changes of this type of cells under different physiological or pathological conditions.

Description

Technical Field

[0001] The invention belongs to the field of brown fat tissue subcellular structure observation, and more specifically, relates to a new fixation method for brown fat transmission electron microscope samples. Background Art

[0002] Brown adipocytes are rich in mitochondria and dispersed small lipid droplets, and can consume fatty acids and glucose through non-shivering heat production. When exposed to cold, the number of mitochondria in these cells increases, the density of mitochondrial cristae increases, and the turnover of lipid droplets increases. Observing the morphology and distribution of mitochondria and lipid droplets at the ultrastructural level is an important indicator for evaluating the metabolic state of brown fat.

[0003] The conventional electron microscopy sample fixation method currently has the following fixative configurations: the pre-fixative is a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde; the post-fixative is 1% osmium tetroxide, both of which are prepared in 0.1 mol / L phosphate buffer (PB). Sample processing: After the mouse is anesthetized, the interscapular brown adipose tissue is separated (the surrounding white adipose tissue is removed), and the tissue is cut into 1 mm 3 The small pieces were then transferred to a centrifuge tube filled with the pre-fixative and fixed overnight on a shaker with 360° continuous rotation at 4°C; the samples were thoroughly rinsed with 0.1 mol / L PB and fixed with the post-fixative at room temperature for 2 h.

[0004] However, it was found that the ultrastructure of brown adipocytes prepared by the current conventional transmission electron microscopy fixation method was not well preserved, the mitochondrial cristae were not clear, and the lipid droplets appeared vacuolar, which could not meet the research needs. Summary of the invention

[0005] To this end, a series of improvements were made to the conventional fixation method, mainly including increasing the concentration of paraformaldehyde in the pre-fixative from the original 2% to 4%; replacing the buffer of the post-fixative osmium acid from the original phosphate buffer to imidazole buffer; and using a fixation method that combines cardiac perfusion and immersion. The new fixation method obtained well-preserved mitochondrial and lipid droplet ultrastructures, and successfully observed the adaptive changes of mitochondrial cristae and lipid droplets in brown adipocytes under cold stimulation conditions, laying the foundation for subsequent research on the structure and functional regulation of brown fat.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] 1. Reagent Configuration

[0008] 1) Preparation of phosphate buffered saline for perfusion: Add 0.625 g NaNO2 and 200 μl heparin (the concentration of heparin stock solution is 6250 units / mL) to 125 ml PBS. Heparin has an anticoagulant effect and NaNO2 has a vasodilator effect.

[0009] 2) The pre-fixative was a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde (prepared with 0.1 mol / L phosphate buffer); the post-fixative was 2% osmium tetroxide prepared with imidazole buffer, the preparation method was: 4% osmium tetroxide: 0.2 mol / L phosphate buffer: 0.2 mol / L imidazole = 3:2:1, pH 7.5.

[0010] 2. Perfusion Fixation

[0011] After the mouse is anesthetized, the right atrial appendage is cut open, and the phosphate buffer for perfusion preheated at 37°C is uniformly perfused from the left ventricle to quickly flush out the blood (about 10mL of phosphate buffer for perfusion is required, which must be completed within 30s), and then the heart is immediately perfused and fixed with 8-12mL of the pre-fixative precooled at 4°C. The entire perfusion is completed within 2min. After perfusion fixation, the brown adipose tissue is separated, and the sample is placed in the pre-fixative and fixed at room temperature for 1.8-2.2h according to the above conventional method; then fixed in the post-fixative at room temperature for 0.8-1.2h.

[0012] Different from the prior art, the above technical solution has the following beneficial effects: Compared with conventional methods, the structure of mitochondrial cristae and other membrane organelles of brown fat cells prepared by the new fixation method proposed in the present invention is more complete; lipid droplets appear as round structures with high electron density. The new fixation method was used to observe the increase in mitochondrial cristae density and lipid droplets in brown fat cells after cold adaptation. The new fixation method can better preserve the ultrastructure of organelles in brown fat cells, especially mitochondria and lipid droplets, and can be used to study the ultrastructural changes that occur in this type of cells under different physiological or pathological conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the gross and light microscopic morphology of the brown adipose tissue between the scapula of mice;

[0014] Figure 2 Representative images of the ultrastructure of brown adipocytes before and after the fixation method was improved;

[0015] Figure 3 Schematic diagram of the ultrastructural remodeling of mouse brown adipocytes under cold training. DETAILED DESCRIPTION

[0016] In order to explain the technical content, structural features, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0017] The C57 / B6 mice used in the experiment were bred in the laboratory, and the stock mice were from Nanjing University-Nanjing Institute of Biomedicine (No. #J000664). The mice were bred and raised in a specific pathogen free (SPF) animal room, given a normal standard diet, and kept at 24°C with a 12-h light / 12-h dark day and night cycle. When the mice were 2 months old, they were transferred to a cold light source constant temperature incubator (Ningbo Jiangnan Instrument Factory, DGXM-508A) for feeding, and the incubator was used to accurately control the feeding environment temperature at 30°C. After the mice were raised at 30°C for 1 month, the temperature of the feeding box was reduced to 4°C by gradient cooling method for cold induction, and they were raised at 4°C for 49 hours. All experimental animal operations were in accordance with the requirements of the Naval Medical University Laboratory Animal Operation Guide (Animal Qualification Certificate No.: SYXK (Shanghai) 2017-0004).

[0018] Conventional fixation method

[0019] Fixative preparation: The pre-fixative was a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde; the post-fixative was 1% osmium tetroxide, both prepared in 0.1 mol / L phosphate buffer (PB). Sample processing: After the mice were anesthetized, the interscapular brown adipose tissue was isolated (the surrounding white adipose tissue was removed) and the tissue was cut into 1 mm 3 The small pieces were then transferred to a centrifuge tube filled with the pre-fixative and fixed overnight at 4°C with 360° continuous rotation on a shaker; the samples were thoroughly rinsed with 0.1 mol / L PB and fixed with the post-fixative at room temperature for 2 h.

[0020] A novel fixation method for brown fat transmission electron microscopy samples in a preferred embodiment of the present invention comprises the following specific steps:

[0021] 1. Reagent configuration

[0022] 1) Preparation of phosphate buffered saline for perfusion: Add 0.625 g NaNO2 and 200 μl heparin (the concentration of heparin stock solution is 6250 U / mL) to 125 ml PBS (phosphate buffered saline, abbreviated as PBS). Heparin has an anticoagulant effect and NaNO2 has a vasodilator effect.

[0023] 2) The pre-fixative was a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde, which was prepared by adding paraformaldehyde and glutaraldehyde into 0.1 mol / L phosphate buffer solution (abbreviated as PB) to form a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde; the post-fixative was 2% osmium tetroxide prepared with imidazole buffer solution, the preparation method was: 4% osmium tetroxide: 0.2 mol / L phosphate buffer solution: 0.2 mol / L imidazole = 3:2:1, pH 7.5.

[0024] 2. Perfusion fixation

[0025] After the mice were anesthetized, the right atrial appendage was cut open, and phosphate buffer for perfusion preheated at 37°C was uniformly perfused from the left ventricle. The perfusion pressure was set to achieve moderate filling of the liver, and the blood was quickly flushed out (about 10 ml of phosphate buffer for perfusion was required, and it was completed within 30 seconds). Then, 10 ml of pre-fixative precooled at 4°C was immediately used for cardiac perfusion fixation. The entire perfusion was completed within 2 minutes. After perfusion fixation, the brown adipose tissue was separated, and the sample was immersed in the pre-fixative at room temperature for 2 hours; then fixed in the post-fixative at room temperature for 1 hour.

[0026] 3. Sample processing after fixation

[0027] After the samples were fixed, they were dehydrated with gradient ethanol and acetone, infiltrated with embedding agents, and embedded. The ultrathin sections were 70 nm thick, and three tissue blocks were cut for each sample. The sections of the conventional fixation method required conventional lead and uranium staining, while the sections of the new fixation method did not require uranium and lead staining and were directly observed under a transmission electron microscope (Hitachi, H7650).

[0028] 4. Quantification of Mitochondrial Cristae

[0029] Electron microscopic photos magnified 3000 times and 5000 times can be used for quantitative analysis of mitochondrial cristae. The sum of the cross-sectional area of ​​mitochondria and the length of cristae was measured using ImageJ software. The formula for calculating cristae density is: cristae density = cristae length sum / mitochondrial cross-sectional area. Three tissues were observed for each sample, and 3-6 cells were observed in each tissue section. Only mitochondria with clear boundaries and all cristae membranes were counted. The independent sample Student-t test (t-test) was used for statistical comparison between the thermoneutral group and the cold treatment group, and the data were presented as mean ± standard error. When P < 0.05, the difference was considered statistically significant.

[0030] 5. Methods for Quantification of Lipid Droplets

[0031] Low-magnification (300x) electron microscopy images were used for quantitative analysis of lipid droplets. The area of ​​the image was measured using ImageJ (V 1.53k), and the number of lipid droplets in the image was measured using the multi-point tool (approximately 7-10 cell sections per image). The lipid droplets per 1000 μm were calculated. 2 The number of lipid droplets per area. Three tissues were observed for each sample, and 1-2 images were counted for each tissue. The independent sample Student-t test (t-test) was used for statistical comparison between the thermoneutral group and the cold treatment group, and the data were presented as mean ± standard error. When P < 0.05, the difference was considered statistically significant.

[0032] See also Figure 1The figure shows the general appearance and microscopic morphology of brown adipose tissue between the scapula of mice. The brown adipose tissue between the scapula of adult mice is generally brown and can be easily distinguished from the surrounding white adipose tissue ( Figure 1 A), light microscopy shows that brown fat cells stained with HE are rich in vacuolar lipid droplet structures ( Figure 1 B).

[0033] See also Figure 2 The following is a representative image of the ultrastructure of brown fat cells. Figure 2 AC. Representative ultrastructural images of brown adipocytes prepared by conventional fixation method; DF. Representative ultrastructural images of brown adipocytes prepared by new fixation method (D: showing the rich mitochondria (Mt) and high-electron-density lipid droplets (LD) of varying sizes in brown adipocytes under low magnification, E: showing the different electron staining of the outer and inner layers of lipid droplets and abundant glycogen granules (GC) in brown adipocytes under high magnification, F: showing the mitochondria, cell membrane (CM) and vesicles (VS) in brown adipocytes under high magnification; scale bars: A and D = 2 μm, B and E = 1 μm, C and F = 500 nm).

[0034] In order to further verify the applicability of the new fixation method, the ultrastructural changes of brown adipocytes during cold induction were observed using the new fixation method. Figure 3 The figure shows the ultrastructural remodeling of brown fat cells in mice under cold training. It was found that after the mice were transferred from a thermoneutral (30°C) environment to a 4°C cold adaptation environment for 49 hours, the lipid droplet morphology in brown fat cells changed from "large lipid droplets" to more "small lipid droplets" ( Figure 3 A, B, D, E), quantitative analysis results showed that the number of lipid droplets per unit area increased significantly ( Figure 3 G); at the same time, the density of mitochondrial cristae also increased significantly ( Figure 3 C, F, H), suggesting that cold stimulation induces ultrastructural "remodeling" of brown adipocytes.

[0035] The new fixation method takes advantage of the rapidity and uniformity of perfusion fixation and improves the fixation effect. The perfusion pressure and time need to be controlled during perfusion fixation. When the perfusion pressure is appropriate, the liver can maintain its normal size. If the liver is significantly enlarged, it indicates that the perfusion pressure is too high. During the period of perfusion of PBS, the tissue is already in a state of ischemia and hypoxia, so the time must be strictly limited. Most of the blood should be flushed out of the body in the shortest time (30s) and then the pre-fixative should be immediately perfused. A mixture of 4% paraformaldehyde and 2.5% glutaraldehyde was selected as the pre-fixative, and the fast penetration speed of paraformaldehyde and the stronger cross-linking properties of glutaraldehyde were used to better preserve the fine structure of the cells.

[0036] The concentration of osmium acid in the new fixative is increased by 1 times, and imidazole is used as a buffer component. Osmium acid is a strong oxidant that can form crosslinks with proteins to stabilize proteins. More importantly, it can combine with unsaturated fatty acid chains to form complexes, and is the only fixative that can fix lipids. However, the fixation effect of osmium acid configured with conventional buffer on lipids is not good enough, and the lipid components in the lipid droplets are easily extracted in the subsequent dehydration link, causing the lipid droplets to present cavitation. Imidazole can significantly enhance the mutual combination of osmium acid and unsaturated fatty acids such as linolenic acid, so that structures such as lipid droplets and biomembranes can be combined with more osmium acid, so that these fine structures can be better preserved. At the same time, since these ultrastructures are combined with more high-electron-density metal osmium, the contrast under the electron microscope is increased, so the present invention can observe mitochondria and other membrane organelles with strong contrast and lipid droplet structures with high electron density without double staining with uranium and lead. But if you need to observe structures such as the nucleus, you can use uranyl acetate staining to enhance the contrast.

[0037] The new fixation method can well preserve the membrane organelles such as mitochondria and lipid droplet structures in brown fat cells, so it can be used to study the ultrastructural changes that occur in the cells under different physiological or pathological conditions. The new fixation method used in the present invention successfully observed the ultrastructural "remodeling" phenomena such as increased density of mitochondrial cristae after cold induction, and changes in the size and number of lipid droplets, and the image quality can meet the qualitative and quantitative requirements of ultrastructural analysis. In addition, the present invention can also be applied to the study of possible ultrastructural changes (such as whitening of brown fat) that occur in brown fat cells under pathological conditions such as metabolic homeostasis imbalance. It is speculated that the new fixation method is also suitable for studying other cells rich in mitochondria and lipid components, such as hepatocytes. Whether this method has advantages for cell types with less mitochondrial and lipid component content remains to be further experimentally verified.

[0038] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "include..." or "comprise..." do not exclude the existence of other elements in the process, method, article or terminal device including the elements. In addition, in this article, "greater than", "less than", "exceed" and the like are understood to exclude the number itself; "above", "below", "within" and the like are understood to include the number itself.

[0039] Although the above embodiments have been described, once those skilled in the art know the basic creative concepts, they can make additional changes and modifications to these embodiments. Therefore, the above description is only an embodiment of the present invention and does not limit the patent protection scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the specification and drawings of the present invention, or directly or indirectly used in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A novel fixation method for brown fat transmission electron microscopy samples, characterized in that: The method comprises the following steps: after the rodent is anesthetized, the right atrial appendage is cut open, and 8 to 12 ml of a phosphate buffered saline solution preheated at 37°C is uniformly perfused from the left ventricle within 30 seconds to quickly flush out the blood; then, 8 to 12 ml of a pre-fixative precooled at 4°C is immediately used for cardiac perfusion fixation, and the entire perfusion is completed within 2 minutes; after perfusion fixation, the brown fat tissue is separated and the tissue is cut into 1 mm 3 The small pieces were then placed in the pre-fixant and fixed at room temperature for 1.8 to 2.2 hours. After the pre-fixation, they were rinsed with 0.1 mol / L phosphate buffer for 3 times, 10 to 15 minutes each time, and rinsed with 0.1 mol / L imidazole for 2 times, 10 to 15 minutes each time. Then they were fixed in the post-fixant at room temperature for 0.8 to 1.2 hours. The phosphate buffered saline solution for perfusion is prepared by adding 0.625 g of NaNO2 and 200 μl of heparin with a concentration of 6250 U / ml to 125 ml of phosphate buffered saline solution; The pre-fixative is a mixture containing 4% paraformaldehyde and 2.5% glutaraldehyde; The post-fixative is prepared in the following manner: 4% osmium tetroxide: 0.2 mol / L phosphate buffer: 0.2 mol / L imidazole = 3:2:1, pH 7.5.

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