Paraffin section elastic fiber overdyeing basic fuchsin composite dyeing kit and dyeing method thereof

Abstract

The invention provides a paraffin section elastic fiber overdyeing basic fuchsin composite dyeing kit and a dyeing method thereof, and belongs to the technical field of pathology, a dyeing reagent in the dyeing kit is prepared from the following raw materials in percentage by weight: 4% of basic fuchsin, 50% of AR pure methanol, 0.3% of thymol, 0.6% of chloral hydrate, 1.5% of phenol and 43.6% of pure water. The dyeing step is simplified, the dyeing time is shortened, the experiment cost is reduced, the pathological diagnosis efficiency is improved, the dyed elastic fiber is specifically red, the display is clear, the background is clean, the stability and repeatability of the dyeing effect are improved, the dyeing error is reduced, the dyeing specificity is improved, non-specific dyeing is reduced, and observation and diagnosis are facilitated.

Description

Technical Field

[0001] This invention relates to the field of pathology technology, and in particular to a kit for basic fuchsin composite staining of paraffin sections with elastic fibers and a staining method thereof. Background Technology

[0002] Elastic fibers are an important component of connective tissue, widely distributed throughout various organs of the human body, such as blood vessels, skin, and lungs. In pathological diagnosis, changes in the morphology, distribution, and quantity of elastic fibers are of great significance for the diagnosis and differential diagnosis of various diseases, such as: vascular diseases: atherosclerosis, aneurysm, vasculitis, etc.; lung diseases: emphysema, pulmonary fibrosis, etc.; skin diseases: cutis laxity, pseudoxanthoma of elastic fibers, etc. The significance of elastic fiber staining in lung cancer and colorectal cancer is also discussed.

[0003] Currently, the following methods are mainly used for staining elastic fibers in pathological tissue sections: Verhoeff staining: This method utilizes iron hematoxylin to bind with elastin in elastic fibers, making the fibers appear black. The staining effect is stable, but the procedure is cumbersome and the staining time is long. Weigert staining: This method utilizes resorcinol fuchsin to bind with elastic fibers, making them appear red. The staining time is shorter, but the staining effect is easily affected by the pH of the fixative and staining solution. Orcein staining: This method utilizes lichen red to bind with elastic fibers, making them appear brownish-red. The procedure is simple, but the staining effect is unstable and prone to fading. Victoria Blue staining: This is a commonly used staining method for displaying elastic fibers in tissue sections. This method utilizes a specific dye to bind with elastin in elastic fibers, making the fibers appear clearly blue under a microscope, facilitating pathological observation and diagnosis. However, its staining time is too long (requiring overnight soaking), which makes it difficult to guarantee diagnostic timeliness.

[0004] Existing methods for staining elastic fibers have the following drawbacks: Cumbersome staining procedures: Some methods involve complex procedures and lengthy staining times, hindering the efficiency of pathological diagnosis. Unstable staining results: Staining results are easily affected by various factors, such as tissue fixative, pH of the staining solution, and staining time, leading to poor reproducibility. Low specificity: Some staining methods lack specificity for elastic fibers, easily resulting in non-specific staining with other tissue components, affecting observation and diagnosis. Summary of the Invention

[0005] The purpose of this invention is to provide a basic fuchsin composite staining kit and method for staining paraffin sections with elastic fibers, thereby solving the technical problems mentioned in the background art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A basic fuchsin composite staining kit for elastic fibers in paraffin sections, wherein the staining reagent in the kit is prepared from the following raw materials in the following proportions: 4-6% basic fuchsin, 50-52% pure methanol (AR), 0.2-0.4% thymol, 0.4-0.8% trichloroacetaldehyde hydrate, 1.5-1.7% phenol, and 41-45% pure water.

[0008] Furthermore, the staining reagents in the staining kit are prepared from the following raw materials in the following proportions: 4% basic fuchsin, 50% AR pure methanol, 0.3% thymol, 0.6% trichloroacetaldehyde hydrate, 1.5% phenol, and 43.6% pure water.

[0009] Xylene (analytical grade, 100% used alone for dewaxing and clearing paraffin sections), pure ethanol (analytical grade, 100% used alone for dexylating and dehydrating paraffin sections), the new formula staining reagent consists of: (basic fuchsin 4%, AR pure methanol 50%, thymol 0.3%, trichloroacetaldehyde hydrate 0.6%, phenol 1.5% and pure water 43.6%), 1% hydrochloric acid alcohol (1ml hydrochloric acid + 99ml 70% alcohol, 100% used alone for section differentiation), and hematoxylin (100% used alone for nuclear staining of sections).

[0010] A staining method using a basic fuchsin compound staining kit for elastic fiber staining of paraffin sections, the method comprising the following steps:

[0011] Step 1: Prepare the staining reagent;

[0012] Step 2: Process the tissue sections to obtain paraffin sections;

[0013] Step 3: Drip or immerse the prepared staining reagent onto the paraffin sections;

[0014] Step 4: Perform the first differentiation of paraffin sections with hydrochloric acid and alcohol, lightly stain the cell nuclei with hematoxylin, and then perform a second differentiation treatment with hydrochloric acid and alcohol.

[0015] Step 5: Apply transparency.

[0016] Step 6: Mounting: After staining, wipe away excess liquid around the slide, add an appropriate amount of neutral resin to the slide and mount it. Observe and photograph it under a microscope.

[0017] Furthermore, the specific process of step 1 is as follows:

[0018] Step 1.1: Dissolve 4g of basic fuchsin in 50mL of AR pure methanol and mix well to prepare solution A;

[0019] Step 1.2: Place 1.5g of phenol in a 70° oven and let it melt into a liquid. Then mix it with 41.4ml of pure water, add 0.3g of thymol and 0.6g of hydrated trichloroacetaldehyde and mix well to prepare solution B.

[0020] Step 1.3: After mixing solution A and solution B, filter the mixture with filter paper and store it in a 4°C refrigerator.

[0021] Further, the specific process of step 2 is as follows: First, bake the tissue sections in a 70°C constant temperature oven for 25 minutes. Place the paraffin sections in the section holder and immerse them in the first xylene bath for 10 minutes. Take out the sections and immerse them in the second xylene bath for 10 minutes. Immerse all the samples in pure ethanol for 10 minutes. Take out the sections and immerse them in pure ethanol for 5 minutes. Immerse all the samples in 95% ethanol for 5 minutes. Immerse all the samples in 85% ethanol for 5 minutes. Immerse all the samples in 70% ethanol for 5 minutes. Finally, rinse with tap water for 5 minutes.

[0022] Furthermore, the specific process of step 3 is as follows: the prepared alkaline fuchsin composite staining solution is used to drip or immerse the paraffin sections for 10 minutes, followed by rinsing with tap water for 1 minute.

[0023] Further, the specific process of step 4 is as follows: differentiation with 1% hydrochloric acid alcohol for 5 seconds, rinsing with tap water for 1 minute, light staining of cell nuclei with Mayer hematoxylin solution for 30 seconds, differentiation with 1% hydrochloric acid alcohol for 1 second.

[0024] Lithium carbonate blueing solution blues in 10 seconds, then rinse with tap water for 10 seconds.

[0025] Further, the specific process of step 5 is as follows: after drying the slices in a 50°C oven, they are placed in xylene twice for 5 minutes each time for clearing treatment.

[0026] The present invention, by adopting the above-described technical solution, has the following beneficial effects:

[0027] (1) This invention simplifies the staining steps, shortens the staining time, reduces experimental costs, improves the efficiency of pathological diagnosis, and the stained elastic fibers are a specific red color, which is clearly visible and has a clean background. It improves the stability and repeatability of the staining effect, reduces staining errors, improves staining specificity, reduces non-specific staining, and facilitates observation and diagnosis.

[0028] (2) This kit is simple and quick to use, and can be used to stain elastic fibers of lung and lung cancer, intestine and colorectal cancer tissues on the same slide. The basic fuchsin composite staining reagent for paraffin section elastic fibers of this invention reduces tissue preparation steps, reduces staining time, lowers reagent cost, increases stability, and provides direct comparison of experimental results. Compared with the traditional Victoria blue staining method, it significantly shortens staining time (no oxidation or overnight incubation required), and makes the elastic fibers exhibit a specific red color, making it particularly suitable for differentiating the degree of invasion in lung and colorectal cancer. This invention also provides an application of the basic fuchsin composite staining kit for paraffin section elastic fibers and its staining method. Attached Figure Description

[0029] Figure 1 This is a stained section image of the present invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are merely to provide the reader with a thorough understanding of one or more aspects of the present invention, and these aspects of the invention can be implemented even without these specific details.

[0031] A basic fuchsin composite staining kit for elastic fibers in paraffin sections is provided. The staining reagent in the kit is prepared from the following raw materials in the following proportions: 4% basic fuchsin, 50% AR pure methanol, 0.3% thymol, 0.6% trichloroacetaldehyde hydrate, 1.5% phenol, and 43.6% pure water.

[0032] A staining method using a basic fuchsin compound staining kit for elastic fiber staining of paraffin sections, comprising the following steps:

[0033] Step 1: Preparation of Basic Fuchsin Reagent: ① Dissolve 4g of basic fuchsin in 50mL of pure methanol (AR grade) and mix well to prepare solution A. ② Place 1.5g of phenol crystals in a 70°C oven until the phenol crystals dissolve into a liquid, then mix with 41.4ml of pure water. Add 0.3g of thymol and 0.6g of trichloroacetaldehyde hydrate and mix well to prepare solution B. ③ After mixing solutions A and B, filter through filter paper and store in a 4°C refrigerator.

[0034] Step 2: Staining: ① Before dewaxing, bake the tissue sections in a 70°C oven for 25 minutes. Place the paraffin sections in a slide holder and immerse them in the first xylene bath for 10 minutes. Remove the sections and immerse them in the second xylene bath for 10 minutes. Immerse all samples in pure ethanol for 10 minutes. Remove the sections and immerse them in pure ethanol for 5 minutes. Immerse all samples in 95% ethanol for 5 minutes. Immerse all samples in 85% ethanol for 5 minutes. Immerse all samples in 70% ethanol for 5 minutes. Finally, rinse with tap water for 5 minutes. This method simplifies the oxidation-reduction process using potassium permanganate and oxalic acid solutions (compared to the traditional Victoria Blue method, this method simplifies this step).

[0035] Step 3: Drip or immerse the paraffin sections in the prepared alkaline fuchsin compound stain for 10 minutes. (This significantly shortens the staining time compared to traditional Victoria blue stain, which requires overnight staining.) Rinse with tap water for 1 minute.

[0036] Step 4: Differentiate with 1% hydrochloric acid alcohol for 5 seconds, then rinse with tap water for 1 minute.

[0037] Step 5: Lightly stain the cell nuclei with Mayer hematoxylin solution for 30 seconds.

[0038] Step 6: Differentiate with 1% hydrochloric acid alcohol for 1 second.

[0039] Step 7: Use lithium carbonate blueing solution to blue for 10 seconds, then rinse with tap water for 10 seconds.

[0040] Step 8: After drying the slices in a 50°C oven, immerse them in xylene twice for 5 minutes each time to achieve transparency.

[0041] 9. Mounting: The stained slide, such as... Figure 1 As shown, wipe away excess liquid around the slide, add an appropriate amount of neutral resin to the slide for mounting, and observe and photograph it under a microscope.

[0042] This method achieves the following: assisted diagnosis and staging: elastic fiber staining can clearly show the depth and extent of tumor invasion, providing an important basis for tumor staging.

[0043] Prognostic assessment: The degree of damage to elastic fibers is closely related to the invasiveness and metastatic risk of tumors, and staining results can provide a reference for prognostic assessment.

[0044] Treatment guidance: The results of elastic fiber staining can provide important information for the formulation of surgical plans and postoperative treatment.

[0045] This kit offers a simple and quick staining process, suitable for staining elastic fibers in lung and lung cancer, intestine and colorectal cancer tissues on the same slide. The basic fuchsin composite staining method for paraffin-embedded elastic fibers reduces tissue preparation steps, minimizes staining time, lowers reagent cost, and increases stability. It provides direct comparative results and significantly shortens staining time compared to traditional Victoria blue staining (no oxidation or overnight incubation required), resulting in a specific red color in the elastic fibers. This method is particularly suitable for differentiating the degree of invasion in lung and colorectal cancer. This invention also provides an application of the basic fuchsin composite staining kit for paraffin-embedded elastic fibers and its staining method.

[0046] Matters not covered in this invention are common knowledge.

[0047] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for basic fuchsin composite staining of paraffin sections with elastic fibers, characterized in that: The staining reagents in the staining kit are prepared from the following raw materials in the following proportions: 4-6% basic fuchsin, 50-52% AR pure methanol, 0.2-0.4% thymol, 0.4-0.8% trichloroacetaldehyde hydrate, 1.5-1.7% phenol, and 41-45% pure water.

2. The paraffin section elastic fiber overlay basic fuchsin composite staining kit according to claim 1, characterized in that: The staining reagents in the staining kit are prepared from the following raw materials in the following proportions: 4% basic fuchsin, 50% AR pure methanol, 0.3% thymol, 0.6% trichloroacetaldehyde hydrate, 1.5% phenol, and 43.6% pure water.

3. The staining method of the basic fuchsin composite staining kit for elastic fibers in paraffin sections according to claim 1, characterized in that: The method includes the following steps: Step 1: Prepare the staining reagent; Step 2: Process the tissue sections to obtain paraffin sections; Step 3: Drip or immerse the prepared staining reagent onto the paraffin sections; Step 4: Perform the first differentiation of paraffin sections with hydrochloric acid and alcohol, lightly stain the cell nuclei with hematoxylin, and then perform a second differentiation treatment with hydrochloric acid and alcohol. Step 5: Apply transparency. Step 6: Mounting: After staining, wipe away excess liquid around the slide, add an appropriate amount of neutral resin to the slide and mount it. Observe and photograph it under a microscope.

4. The staining method of the basic fuchsin composite staining kit for elastic fibers in paraffin sections according to claim 3, characterized in that: The specific process of step 1 is as follows: Step 1.1: Dissolve 4g of basic fuchsin in 50mL of AR pure methanol and mix well to prepare solution A; Step 1.2: Place 1.5g of phenol in a 70° oven and let it melt into a liquid. Then mix it with 41.4ml of pure water, add 0.3g of thymol and 0.6g of hydrated trichloroacetaldehyde and mix well to prepare solution B. Step 1.3: After mixing solution A and solution B, filter the mixture with filter paper and store it in a 4°C refrigerator.

5. The staining method of the basic fuchsin composite staining kit for elastic fibers in paraffin sections according to claim 3, characterized in that: The specific process of step 2 is as follows: First, bake the tissue sections in a 70°C constant temperature oven for 25 minutes. Place the paraffin sections in the section holder and immerse them in the first xylene bath for 10 minutes. Take out the sections and immerse them in the second xylene bath for 10 minutes. Immerse all the samples in pure ethanol for 10 minutes. Take out the sections and immerse them in pure ethanol for 5 minutes. Immerse all the samples in 95% ethanol for 5 minutes. Immerse all the samples in 85% ethanol for 5 minutes. Immerse all the samples in 70% ethanol for 5 minutes. Finally, rinse with tap water for 5 minutes.

6. The staining method of the basic fuchsin composite staining kit for elastic fibers in paraffin sections according to claim 3, characterized in that: The specific process of step 3 is as follows: Drip or immerse the paraffin sections in the prepared alkaline fuchsin composite dye solution for 10 minutes, and then rinse with tap water for 1 minute.

7. The staining method of the basic fuchsin composite staining kit for elastic fiber overlay staining of paraffin sections according to claim 3, characterized in that: The specific process of step 4 is as follows: differentiation with 1% hydrochloric acid alcohol for 5 seconds, rinsing with tap water for 1 minute, light staining of cell nuclei with Mayer hematoxylin solution for 30 seconds, differentiation with 1% hydrochloric acid alcohol for 1 second, re-blueing with lithium carbonate solution for 10 seconds, and rinsing with tap water for 10 seconds.

8. The staining method of the basic fuchsin composite staining kit for elastic fibers in paraffin sections according to claim 3, characterized in that: The specific process of step 5 is as follows: After drying the slices in a 50°C oven, immerse them in xylene twice for 5 minutes each time to achieve transparency.

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