A method for paraffin sectioning of iris cells

Abstract

This invention proposes a method for paraffin sectioning of iris cells. In the sampling and fixation step, phenol and formaldehyde are used as the tissue fixative, with a phenol volume concentration of 1-2% and a formaldehyde mass concentration of 6-8%. This invention solves the problem of insufficient fixation of iris cells obtained by traditional fixation methods, yielding clearly morphologically distinct iris cell tissue sections, which is of great significance for the identification of iris cell species and histological analysis.

Description

Technical Field

[0001] This invention relates to the field of cell staining technology, and in particular to a method for paraffin sectioning of iris cells. Background Technology

[0002] Body color is one of the important traits of fish, and it is also the result of long-term natural selection in the process of fish adapting to their environment. The body color and patterns of fish, such as markings, are formed by the types and numbers of pigment cells distributed in the skin, the state of the pigment granules contained in the pigment cells, and the reflective power of the crystalline reflective plates in the iridocytes. Currently, five types of pigment cells have been found in fish, concentrated in the dermis of the skin tissue: melanin cells, erythrophores, xanthophore cells, canophores, and iridocytes. As an important type of pigment cell in fish, iridocytes participate in many physiological processes through structural coloration, such as increasing salience (e.g., enhancing skin reflectivity, warning coloration, and nuptial coloration), camouflage, thermoregulation mechanisms, rapid changes in body color, and the composition of the iris of the eye. Iridocytes in fish can be divided into two types based on cell size: S-type and L-type iridocytes. The leopard-spotted spiny perch (Plectropomus leopardus), also known as the spotted spiny perch, is a species belonging to the order Perciformes, family Serranidae, subfamily Plectropinae, genus Plectropomus, and species Plectropomus leopardus. It is distributed in tropical or subtropical waters of the Indo-Pacific region, concentrated along the edges of coral reefs in the South China Sea, East China Sea, and western Pacific. It exhibits a complex social structure and diverse color variations, including dark brown, brown, and red. As an important economic fish species in my country, the leopard-spotted spiny perch has developed a unique body coloration through long-term natural selection to adapt to its environment and facilitate population exchange: a bright red body with blue spots distributed on the skin tissue except for the ventral side. Body color is an important economic trait of the leopard-spotted spiny perch. This trait is mainly related to the type, number, and state of pigment cells distributed in the skin tissue. The mechanisms underlying the formation and regulation of body color in the leopard-gill sea bass remain unclear. Domestic research on body color in this sea bass primarily focuses on melanocytes and erythrocytes, while research on iris cells is relatively scarce. This lack of research on iris cells is partly due to their colorless nature and the presence of a unique guanine crystal structure. Therefore, conventional paraffin embedding and HE staining methods often result in iris cell detachment and fragmentation of the entire skin tissue section, making histological observation and analysis of iris cells in the leopard-gill sea bass skin tissue impossible. To understand the functional characteristics of iris cells in leopard-gill sea bass skin tissue and explore their role in body color composition and physiological color changes, histological observation of the leopard-gill sea bass skin is necessary. This includes histological analysis of the types, morphological characteristics, distribution patterns, and changes in iris cells under different physiological states. Therefore, developing suitable conventional embedding and staining methods for iris cells in leopard-gill sea bass is a primary challenge in exploring the role of iris cells in the genetic control and variation of body color in this sea bass. Summary of the Invention

[0003] Therefore, the purpose of this invention is to propose a paraffin sectioning method for iris cells, which solves the problems of insufficient fixation of iris cells and incomplete tissue sections in the leopard gill spiny perch. This method can obtain iris cell tissue sections with clear and complete morphological characteristics, laying a good foundation for exploring the role of iris cells in body color regulation.

[0004] The technical solution of this invention is implemented as follows:

[0005] A method for paraffin sectioning iris cells, wherein during the sampling and fixation step, phenol and formaldehyde are selected as tissue fixatives, wherein the volume concentration of phenol in the tissue fixative is 1-2% and the mass concentration of formaldehyde is 6-8%.

[0006] Accordingly, the fixed temperature is 3–5℃, and the fixed time is 24–36h.

[0007] Accordingly, after sampling and fixation, the process also includes agarose pre-embedding, dehydration and paraffin infiltration, paraffin embedding, tissue sectioning, and section staining.

[0008] The pre-embedding step uses a method for preparing agarose solution, including: first preparing an agarose solution with a mass concentration of 1.8% to 2.2% using ultrapure water, then diluting it with anhydrous ethanol to a mass concentration of 0.8% to 1.2% to obtain the target agarose solution, and using the target agarose solution at a temperature of 40 to 45°C.

[0009] Accordingly, the dehydration and wax impregnation steps include sequential treatment with 70%–75% ethanol solution for 1.5–2.5 h, 80%–85% ethanol solution for 1.5–2.5 h, 90%–95% ethanol solution for 1.5–2.5 h, anhydrous ethanol for 3–5 h, an anhydrous ethanol and xylene mixture for 1.5–2.5 h, xylene for 30–50 min, xylene and paraffin mixture for 0.5–1.5 h, and paraffin for 1.5–2.5 h.

[0010] Accordingly, in the section staining step, xylene, a mixture of xylene and anhydrous ethanol, anhydrous ethanol, 90%–95% ethanol solution (volume concentration), 0.4%–0.6% eosin staining solution (mass concentration), 80%–85% ethanol solution (volume concentration), and 90%–95% ethanol solution (volume concentration) are used for staining treatment.

[0011] Accordingly, during the section staining step, the following treatments were performed sequentially: xylene for 5–7 min, anhydrous ethanol and xylene mixture for 2.5–3.5 min, anhydrous ethanol for 4–6 min, 90%–95% ethanol solution for 1.5–2.5 min, 0.4%–0.6% eosin solution for 0.5–1.5 min, 80%–85% ethanol solution for 1.5–2.5 min, 90%–95% ethanol solution for 1.5–2.5 min, anhydrous ethanol for 5–7 min, anhydrous ethanol and xylene mixture for 2.5–3.5 min, and xylene for 5–7 min.

[0012] Accordingly, when using a mixture of xylene and anhydrous ethanol, the volume ratio of anhydrous ethanol to xylene is 1-2:1-2; when using a mixture of xylene and paraffin, the volume ratio of xylene to paraffin is 1-2:1-2.

[0013] The present invention also provides an application of the above-mentioned paraffin sectioning method for iris cells in the preparation of fish iris cell tissue sections.

[0014] Accordingly, the fish is the leopard-gill spiny perch.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] 1. This invention solves the problem of insufficient fixation of iris cells obtained by traditional fixation methods, and obtains iris cell tissue sections with clear morphology, which is of great significance for the identification of iris cell species and histological analysis.

[0017] 2. This invention solves the problem of incomplete and fragmented skin tissue sections of the Leopard Gill-spined Bass, obtaining morphologically intact skin tissue sections. This is of great significance for the analysis of the distribution of iris cells and other pigment cells in the Leopard Gill-spined Bass, as well as the morphological changes and distribution of iris cells under different physiological conditions. Furthermore, the method of this invention can identify the types of iris cells that were previously missed in the Leopard Gill-spined Bass.

[0018] 3. This invention can be used for histological analysis of iris cells in other fish species, providing a more suitable method for histological research on iris cells and skin in fish, and also promoting the study of the role of iris cells in the regulation of body color in fish. Attached Figure Description

[0019] Figure 1 This is a flowchart of the histological analysis experiment of iris cells of the leopard-gill spiny perch in Example 1 of the present invention.

[0020] Figure 2The images show the staining results of iris cell histology in agarose-paraffin sections of the leopard-gill spiny perch from Example 1 of this invention. Figures A, D, and F show the section observation results under a 40x objective lens, with a scale bar of 50 μm. Figures B, C, E, and G show the observation results of the white box area in the 40x objective lens section observation results under a 100x objective lens, with a scale bar of 20 μm. The green arrows indicate iris cells.

[0021] Figure 3 The image shows the staining results of a conventional paraffin section of skin from the leopard-gill spiny perch, Comparative Example 1 of this invention. In the image, Figures A, B, and C are scale bars of 100 μm, 50 μm, and 20 μm, respectively.

[0022] Figure 4 Figure A shows the staining results obtained in Example 1 and Comparative Example 1 of the present invention. Figure A shows a skin tissue section of Leopard Gill-spined Bass using the agarose-paraffin embedding method established in Example 1 of the present invention, with iris cells marked by green arrows. Figure B shows skin tissue sections of Leopard Gill-spined Bass using the conventional fixation and paraffin embedding methods in Comparative Example 1. Detailed Implementation

[0023] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0024] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0026] In this embodiment of the invention, reagent names I and II are the same reagent, but they are used in two separate portions.

[0027] Example 1

[0028] The assay protocol for iridocellular tissue analysis of 1.5-year-old leopard-gill spiny perch is as follows:

[0029] (1) Preparation of tissue fixative: Heat phenol in a water bath until it melts, and use a pipette to add 1 ml of phenol liquid to 50 ml of 8% formaldehyde solution to obtain tissue fixative. Shake well and set aside.

[0030] (2) Sampling and fixation: The skin tissue sample of the leopard gill spiny perch was gently torn off with 75% alcohol-sterilized dissecting scissors and forceps, washed with PBS buffer, and then placed in tissue fixation solution and fixed at 4°C for 30 h.

[0031] (3) Agarose pre-embedding: First, prepare a 2% agarose solution with ultrapure water, then dilute it with anhydrous ethanol while hot to a 1% agarose solution. Wait for the solution to cool to 40-45℃ before placing it into the fixed skin tissue. After the agarose gel has completely solidified, use a scalpel to trim the embedded tissue into a cube of about 3mm × 3mm.

[0032] (4) Dehydration and paraffin infiltration: The tissue embedded in agarose gel was subjected to gradient dehydration. The dehydration and paraffin infiltration processes were carried out in the order shown in Table 1 below:

[0033] Table 1. Dehydration and wax impregnation process of agarose-paraffin sections

[0034]

[0035]

[0036] (5) Paraffin embedding: After paraffin infiltration, the skin tissue is placed in an embedding machine for tissue embedding. The tissue is placed in a metal embedding frame. Note that when cutting the tissue block transversely, a pestle should be used to carefully flatten the tissue to ensure that the tissue is on the same plane when sectioning later. After the paraffin block solidifies on the cooling stage, trim the paraffin and store it in a -20°C refrigerator for later sectioning.

[0037] (6) Tissue sectioning: Paraffin sectioning of tissue was performed using an automatic roller microtome, with a section thickness of 6–8 μm. The slide spreader was set to a constant temperature of 42°C, and the skin tissue sections were flattened with ultrapure water. The flattened tissue sections were then picked up with a glass slide. The slide dryer was set to 37°C, and the freshly picked-up tissue sections were placed on the slide dryer to dry overnight.

[0038] (7) Staining the sections: Dewax and stain the sections by placing them into a staining jar containing the reagents in Table 2.

[0039] Table 2. Agarose-paraffin section staining process.

[0040]

[0041] (8) Mounting: Take out the xylene slices and lay them flat on the test bench, then mount them with neutral resin.

[0042] (9) Microscopic observation under an optical microscope: Images were taken from skin tissue sections of the leopard gill spiny perch at 200, 400, and 1000x magnification using an optical microscope.

[0043] Comparative Example 1

[0044] Currently, the method for histological analysis of iridocornus iridocornus involves paraffin sections and hematoxylin-eosin (HE) staining to aid in the observation of cell morphology and structure. The specific procedure is as follows:

[0045] (1) Sampling and fixation: The skin tissue samples of leopard gill spiny perch were washed with PBS buffer and then fixed in 4% paraformaldehyde fixative (PFA).

[0046] (2) Dehydration and paraffin infiltration: The fixed tissue is dehydrated in a gradient manner, and the dehydration and paraffin infiltration processes are carried out in the following order as shown in Table 3:

[0047] Table 3. Dehydration and wax impregnation process of ordinary paraffin slices

[0048]

[0049] (3) Tissue embedding: After the skin tissue was immersed in paraffin, it was placed in an embedding machine for tissue embedding and stored at -20°C for later sectioning.

[0050] (4) Tissue sectioning: Paraffin sectioning of tissue was performed using an automatic roller microtome, with a section thickness of 6–8 μm. Skin tissue sections were flattened with ultrapure water and dried overnight on a drying machine.

[0051] (5) Staining: The sections were stained using the hematoxylin-eosin (HE) staining method. The dried sections were dewaxed and stained, and the sections were placed in the staining jars containing the reagents listed in Table 4.

[0052] Table 4. Hematoxylin-eosin staining process

[0053]

[0054]

[0055] (6) Mounting: Take out the xylene slices and lay them flat on the test bench, then mount them with neutral resin.

[0056] (7) Microscopic examination with an optical microscope: Skin tissue sections of the leopard gill spiny perch were selected and images were collected using an optical microscope.

[0057] Depend on Figure 4 Comparative analysis shows that during HE staining, insufficient cell fixation resulted in almost no observable iris cells in the sections. PFA fixative dissolved intracellular reflective plates, and the lack of agarose embedding easily led to tissue damage. Furthermore, cells were prone to tearing during sectioning. Figure 4 B) The staining process is not only cumbersome, but it also easily causes the iris cytoplasm to be stained by nuclear dyes, resulting in blurred observation of the internal structure of the cell.

[0058] There are two types of iris cells: L-type and S-type. Currently, traditional histological analysis methods for leopard-gill spiny perch have only found L-type iris cells. However, the method in Example 1 of this invention can reveal that leopard-gill spiny perch has both L-type and S-type iris cells.

[0059] Comparative Example 2

[0060] The difference between this comparative example and Example 1 is that the tissue fixative was prepared by adding 0.25 ml of phenol liquid to 50 ml of 8% formaldehyde solution to obtain the tissue fixative. Subsequent steps were the same as in Example 1.

[0061] Upon examination, the structure and outline of the iris cells were not clear, and there was iris cell detachment.

[0062] Example 2

[0063] The difference between this embodiment and Embodiment 1 lies in the treatment of the section staining step, as detailed below:

[0064] (1) Preparation of tissue fixative: Heat phenol in a water bath until it melts, and use a pipette to add 1 ml of phenol liquid to 50 ml of 8% formaldehyde solution to obtain tissue fixative. Shake well and set aside.

[0065] (2) Sampling and fixation: The skin tissue sample of the leopard gill spiny perch was gently torn off with 75% alcohol-sterilized dissecting scissors and forceps, washed with PBS buffer, and then placed in tissue fixation solution and fixed at 4°C for 30 h.

[0066] (3) Agarose pre-embedding: First, prepare a 2% agarose solution with ultrapure water, then dilute it with anhydrous ethanol while hot to a 1% agarose solution. Wait for the solution to cool to 40-45℃ before placing it into the fixed skin tissue. After the agarose gel has completely solidified, use a scalpel to trim the embedded tissue into a cube of about 3mm × 3mm.

[0067] (4) Dehydration and paraffin infiltration: The tissue embedded in agarose gel was subjected to gradient dehydration. The dehydration and paraffin infiltration processes were carried out in the order shown in Table 5 below:

[0068] Table 5. Dehydration and wax impregnation process of agarose-paraffin sections

[0069]

[0070]

[0071] (5) Paraffin embedding: After paraffin infiltration, the skin tissue is placed in an embedding machine for tissue embedding. The tissue is placed in a metal embedding frame. Note that when cutting the tissue block transversely, a pestle should be used to carefully flatten the tissue to ensure that the tissue is on the same plane when sectioning later. After the paraffin block solidifies on the cooling stage, trim the paraffin and store it in a -20°C refrigerator for later sectioning.

[0072] (6) Tissue sectioning: Paraffin sectioning of tissue was performed using an automatic roller microtome, with a section thickness of 6–8 μm. The slide spreader was set to a constant temperature of 42°C, and the skin tissue sections were flattened with ultrapure water. The flattened tissue sections were then picked up with a glass slide. The slide dryer was set to 37°C, and the freshly picked-up tissue sections were placed on the slide dryer to dry overnight.

[0073] (7) Staining the sections: Dewax and stain the sections by placing them into a staining jar containing the reagents in Table 6.

[0074] Table 6. Agarose-paraffin section staining process.

[0075]

[0076]

[0077] (8) Mounting: Take out the xylene slices and lay them flat on the test bench, then mount them with neutral resin.

[0078] (9) Microscopic observation under an optical microscope: Images were taken from skin tissue sections of the leopard gill spiny perch at 200, 400, and 1000x magnification using an optical microscope.

[0079] Testing revealed cell fragmentation, incomplete morphology, and significant damage to the internal structure of some iris cells, with noticeable detachment of the sample.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of paraffin sectioning of iridophores of Ctenochaetus strigosus, characterized by, During the sampling and fixation step, phenol and formaldehyde are selected as tissue fixatives, wherein the volume concentration of phenol in the tissue fixative is 1-2% and the mass concentration of formaldehyde is 6-8%. The fixed temperature is 3~5℃, and the fixed time is 24~36h; After sampling and fixation, the process also includes agarose pre-embedding, dehydration and paraffin infiltration, paraffin embedding, tissue sectioning, and section staining. During the section staining process, the following steps were performed sequentially: xylene treatment for 5–7 min, anhydrous ethanol and xylene mixture treatment for 2.5–3.5 min, anhydrous ethanol treatment for 4–6 min, 90%–95% ethanol solution treatment for 1.5–2.5 min, 0.4%–0.6% eosin staining solution treatment for 0.5–1.5 min, 80%–85% ethanol solution treatment for 1.5–2.5 min, 90%–95% ethanol solution treatment for 1.5–2.5 min, anhydrous ethanol treatment for 5–7 min, anhydrous ethanol and xylene mixture treatment for 2.5–3.5 min, and xylene treatment for 5–7 min. The volume ratio of anhydrous ethanol to xylene is 1~2:1~2.

2. The method of paraffin sectioning of caudal fin of Cephalopholis leopardus iridophores according to claim 1, characterized in that, The preparation method of the agarose solution used in the pre-embedding step includes: first, preparing an agarose solution with a mass concentration of 1.8%~2.2% using ultrapure water, and then diluting it with anhydrous ethanol to a mass concentration of 0.8%~1.2% to obtain the target agarose solution. The temperature at which the target agarose solution is used is 40~45℃.

3. The method for paraffin sectioning iris cells of the leopard-gill spiny perch according to claim 1, characterized in that, The dehydration and wax impregnation steps include sequential treatment with 70%~75% ethanol solution for 1.5~2.5 h, 80%~85% ethanol solution for 1.5~2.5 h, 90%~95% ethanol solution for 1.5~2.5 h, anhydrous ethanol for 3~5 h, a mixture of anhydrous ethanol and xylene for 1.5~2.5 h, xylene for 30~50 min, a mixture of xylene and paraffin for 0.5~1.5 h, and paraffin for 1.5~2.5 h.

4. The method for paraffin sectioning iris cells of the leopard-gill spiny perch according to claim 3, characterized in that, When using a mixture of xylene and paraffin, the volume ratio of xylene to paraffin is 1~2:1~2.

5. The application of a paraffin sectioning method using iris cells of the leopard-gill spiny perch according to any one of claims 1 to 4 in the preparation of L-type and S-type iris cell tissue sections of the leopard-gill spiny perch.

Images