Ammonium salt solution, ferric diamine-alcian blue staining solution and applications thereof

Abstract

The present invention relates to ammonium salt solution, high iron diamine alician blue staining solution and its application, belong to the field of medical detection technology. A metal ion chelating agent is added to the ammonium salt solution of the present invention, because the metal ion chelating agent can chelate the metal ions in the solution, reduce the release of oxygen free radicals, suppress and slow down the color change reaction of N, N-dimethyl-p-phenylenediamine dihydrochloride, so as to better protect N, N-dimethyl-m-phenylenediamine dihydrochloride, improve the storage stability of ammonium salt solution, significantly improve the problem of color deepening or blackening when ammonium salt solution is stored for a long time. In addition, when the ammonium salt solution is used for high iron diamine alician blue staining solution, the staining effect of the staining solution in pathological tissue is not affected, and the background is clean and the staining specificity is good.

Description

Technical Field

[0001] The invention relates to an ammonium salt solution, a ferric diamine-alcian blue staining solution and applications thereof, and belongs to the technical field of medical detection. Background Art

[0002] Ferrous diamine-alcian blue staining solution, also known as mucus HID-AB staining solution, is primarily composed of ferrous diamine solution and alcian blue staining solution. It can stain substances such as acidic mucins, proteoglycans, and hyaluronic acid, rendering acidic mucins (sulfated acidic mucins) brownish purple to brownish black and sialic acidic mucins (proteoglycans, hyaluronic acid) blue, thereby distinguishing between sulfated mucins and proteoglycans. Therefore, ferrous diamine-alcian blue staining solution is primarily used to identify sulfated acidic mucins and sialic acidic mucins.

[0003] The staining principle of the ferric diamine-alcian blue stain lies in the fact that the diammonium salt in the ferric diamine salt combines with sulfated acidic mucus substances to form a complex that develops color. When the pH is significantly below 2.5, the sulfate groups in the tissue ionize, becoming negatively charged. This salt bond forms with the cations in the alcian blue, staining tissues containing sulfate groups (such as sulfated mucus substances) and producing a brownish-purple to brownish-black color. At a pH of 2.5, the carboxyl groups in the tissue ionize, becoming negatively charged. This salt bond forms with the cations in the alcian blue, staining tissues containing carboxyl groups (such as proteoglycans / hyaluronic acid and sialic acid mucins, such as epithelial acidic mucins), producing a blue color.

[0004] Ferric diamine solutions include N,N-dimethyl-p-phenylenediamine dihydrochloride solution (HID-A solution), N,N-dimethyl-m-phenylenediamine dihydrochloride solution (HID-B solution), and ferric chloride solution. Both N,N-dimethyl-p-phenylenediamine dihydrochloride and N,N-dimethyl-m-phenylenediamine dihydrochloride are ammonium salts, carrying a positive charge after dissociation. The diammonium salts combine with sulfated acidic mucus substances to form complexes, which develop color. This reaction is very slow, and ferric chloride acts as a catalyst to increase the reaction rate. Ferric chloride oxidizes the diamine salts to form a brown-black cationic chromogen, thereby accelerating staining. It also lowers the pH of the staining solution to 1.4. At this pH, the carboxyl groups on the section cannot bind to the diamine salts, and only the sulfate groups react with the diamine salts to form purple-brown to brown-black complexes. When the N,N-dimethyl-m-phenylenediamine dihydrochloride solution is left for a long time, its color gradually changes from colorless to darker, causing the staining effect of the high-iron diamine-alecian blue staining solution to weaken and causing mixed colors in the stained tissue. Summary of the Invention

[0005] The present invention aims to provide an ammonium salt solution, which can solve the problem that the N,N-dimethyl-m-phenylenediamine dihydrochloride solution in the current ferric diamine-alecian blue staining solution is prone to discoloration when left for a long time, thereby affecting the staining effect of the ferric diamine-alecian blue staining solution and causing mixed colors in the stained tissue.

[0006] The second object of the present invention is to provide a ferrous diamine-alecian blue staining solution.

[0007] The third object of the present invention is to provide an application of ferric diamine-alecian blue staining solution in the differential staining of sulfated acidic mucus substances and sialic acid mucus substances.

[0008] In order to achieve the above object, the technical solution adopted by the ammonium salt solution of the present invention is:

[0009] An ammonium salt solution is mainly prepared from N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water; or the ammonium salt solution is mainly prepared from N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water; the metal ion chelating agent is disodium ethylenediaminetetraacetate.

[0010] The ammonium salt solution of the present invention is added with a metal ion chelating agent. Since the metal ion chelating agent can chelate the metal ions in the solution, the release of oxygen free radicals is reduced, and the color change reaction of N, N-dimethyl-p-phenylenediamine dihydrochloride is suppressed and slowed down, so that N, N-dimethyl-m-phenylenediamine dihydrochloride can be better protected, the storage stability of the ammonium salt solution is improved, and the problem of color deepening or blackening when the ammonium salt solution is significantly improved during long-term preservation is observed. In addition, when the ammonium salt solution is used for high-iron diamine-alecian blue staining solution, the staining effect of the staining solution in pathological tissue is not affected, and the background is clean and the staining specificity is good. And experimental results show that when the metal ion chelating agent is disodium ethylenediaminetetraacetic acid, the storage stability of the ammonium salt solution can be improved and the ammonium salt solution prepared can be used for high-iron diamine-alecian blue staining solution, without affecting the staining effect of the staining solution in pathological tissue.

[0011] Preferably, the amount of the metal ion chelating agent used for each 0.72 g of N,N-dimethyl-m-phenylenediamine dihydrochloride is 5×10 -8 ~5×10 -5 mol.

[0012] Further preferably, when the ammonium salt solution is mainly made of N, N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the amount of the metal ion chelating agent used for each 0.72 g of N, N-dimethyl-m-phenylenediamine dihydrochloride is 5×10 -8mol;

[0013] When the ammonium salt solution is mainly composed of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the amount of the metal ion chelating agent used for each 0.72g of N,N-dimethyl-m-phenylenediamine dihydrochloride is 5×10 -8 ~5×10 -6 The experimental results show that the higher the EDTA concentration, the better. From the application effect, when the ammonium salt solution is mainly composed of N, N-dimethyl-m-phenylenediamine dihydrochloride, metal ion chelating agent and water, the amount of metal ion chelating agent used for every 0.72g of N, N-dimethyl-m-phenylenediamine dihydrochloride is 5×10 -8 mol, or when the ammonium salt solution is mainly composed of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the amount of the metal ion chelating agent used for every 0.72 g of N,N-dimethyl-m-phenylenediamine dihydrochloride is 5×10 -8 ~5×10 -6 When the concentration of disodium EDTA is 0.1 mol, disodium EDTA can better protect N,N-dimethyl-m-phenylenediamine dihydrochloride. The reason may be that disodium EDTA is weakly acidic. The higher the concentration of disodium EDTA added, the more it affects the stability of N,N-dimethyl-m-phenylenediamine dihydrochloride, resulting in a darker color.

[0014] Preferably, when the ammonium salt solution is mainly made of N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the mass ratio of the N,N-dimethyl-m-phenylenediamine dihydrochloride to water is (0.12-0.72):50;

[0015] When the ammonium salt solution is mainly composed of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the mass ratio of the N,N-dimethyl-m-phenylenediamine dihydrochloride, N,N-dimethyl-p-phenylenediamine dihydrochloride and water is (0.12-0.72):(0.02-0.12):50.

[0016] Further preferably, when the ammonium salt solution is mainly composed of N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the mass ratio of the N,N-dimethyl-m-phenylenediamine dihydrochloride to water is 0.72:50;

[0017] When the ammonium salt solution is mainly composed of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the mass ratio of the N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride and water is 0.12:0.72:50.

[0018] The technical solution adopted by the high-iron diamine-alecian blue staining solution of the present invention is:

[0019] A ferric diamine-alcian blue staining solution comprises a ferric diamine solution and an alcian blue staining solution, wherein the ferric diamine solution comprises a ferric chloride solution and the above-mentioned ammonium salt solution; when the ammonium salt solution is mainly made of N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the ferric diamine solution also comprises an N,N-dimethyl-p-phenylenediamine dihydrochloride solution.

[0020] The high-iron diamine-alecian blue staining solution of the present invention has the advantages of stable storage, stable staining effect, good staining specificity and high staining intensity.

[0021] It can be understood that when an ammonium salt solution mainly composed of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelator and water is used in a ferric diamine-alecian blue staining solution, the mass ratio of N,N-dimethyl-m-phenylenediamine dihydrochloride and N,N-dimethyl-p-phenylenediamine dihydrochloride in the ammonium salt solution is 6:1.

[0022] Preferably, the N,N-dimethyl-p-phenylenediamine dihydrochloride solution is prepared by dissolving 0.02 to 0.12 parts by mass of N,N-dimethyl-p-phenylenediamine dihydrochloride in 50 parts by mass of water.

[0023] Preferably, the ferric chloride solution is prepared by dissolving 3.6 to 5.4 parts by mass of ferric chloride in 100 parts by mass of water.

[0024] Preferably, the Alcian blue staining solution is prepared from Alcian blue, a pH adjuster, and water; the mass ratio of Alcian blue to water is 1:100. Preferably, the pH of the Alcian blue staining solution is 2.5. Preferably, the pH adjuster is acetic acid. Preferably, the Alcian blue staining solution is prepared by dissolving Alcian blue in water and then adjusting the pH.

[0025] Preferably, the ferric diamine-alecian blue staining solution further comprises a nuclear fast red staining solution.

[0026] Preferably, the nuclear fast red staining solution is made of aluminum sulfate and nuclear fast red dissolved in water; the mass ratio of the aluminum sulfate, nuclear fast red and water is 5:0.1:100.

[0027] The technical solution adopted by the application of the high iron diamine-alecian blue staining solution of the present invention in the identification and staining of sulfated acidic mucus substances and sialic acid mucus substances is:

[0028] A use of the ferric diamine-alecian blue staining solution in the differential staining of sulfated acidic mucus substances and sialic acid mucus substances.

[0029] The high iron diamine-alecian blue staining solution of the present invention is used for the identification and staining of sulfated acidic mucus substances and sialic acid mucus substances, has the advantages of good specificity and high staining intensity, and can distinguish sulfated acidic mucus substances and sialic acid mucus substances at a high level.

[0030] It is understood that, in the present invention, sulfated acidic mucus substances mainly refer to sulfur mucins and the like; sialic acid mucus substances mainly refer to proteoglycans, hyaluronic acid, epithelial acidic mucins and the like.

[0031] Preferably, the ferric diamine-alecian blue staining solution includes a nuclear fast red staining solution, and the application includes the following steps:

[0032] (1) When the ammonium salt solution in the ferric diamine-alecian blue staining solution is mainly composed of N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the ammonium salt solution in the ferric diamine-alecian blue staining solution, the N,N-dimethyl-p-phenylenediamine dihydrochloride solution and the ferric chloride solution are mixed and applied to the surface of the object to be tested, followed by incubation, and after the incubation is completed, the object is rinsed with water;

[0033] When the ammonium salt solution in the ferric diamine-alecian blue staining solution is mainly made of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the ammonium salt solution in the ferric diamine-alecian blue staining solution and the ferric chloride solution are mixed and applied to the surface of the object to be tested, followed by incubation, and after the incubation is completed, the object is rinsed with water;

[0034] (2) staining the sample obtained in step (1) with the alecian blue staining solution in the ferrous diamine-alecian blue staining solution, and rinsing with water after the staining is completed;

[0035] (3) re-staining the stained sample obtained in step (2) with the nuclear fast red staining solution in the ferric diamine-alecian blue staining solution;

[0036] (4) Observe the color of the analyte after counterstaining to complete the identification of sulfated acidic mucus substances and sialic acid mucus substances.

[0037] It is understandable that if the substance to be identified is located on a certain tissue, the tissue containing the substance to be identified can be sliced, dewaxed, and hydrated first, and then the N,N-dimethyl-p-phenylenediamine dihydrochloride solution, N,N-dimethyl-m-phenylenediamine dihydrochloride solution and ferric chloride solution in the high-iron diamine-alecian blue staining solution are mixed and applied to the surface of the section of the substance to be identified according to the method of step (1), and then stained with alecian blue staining solution, and then counterstained with nuclear fast red staining solution. The counterstained section is then dehydrated and transparent, and then sealed with neutral gum. Finally, the staining results are observed under a microscope. The part with brown-purple to brown-black color is sulfated acidic mucus substance, and the part with blue color is sialic acid mucus substance.

[0038] Preferably, in step (1), the incubation time is 18 to 24 hours. Preferably, in step (1), the incubation is performed at room temperature.

[0039] Preferably, in step (2), the dyeing time is 10 to 20 minutes.

[0040] Preferably, in step (3), the re-staining time is 3 to 5 minutes.

[0041] Preferably, in step (1), when the ammonium salt solution in the high-iron diamine-alecian blue staining solution is mainly composed of N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelator and water, the volume ratio of the ammonium salt solution, N,N-dimethyl-p-phenylenediamine dihydrochloride solution and the ferric chloride solution is 1:1:1; when the ammonium salt solution in the high-iron diamine-alecian blue staining solution is mainly composed of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelator and water, the volume ratio of the ammonium salt solution to the ferric chloride solution is 1:1. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the appearance of the ammonium salt solution of Comparative Example 1 in Experimental Example 1 after being placed at 4°C and 37°C for 2 weeks respectively; wherein, Figure 1 a is the appearance of the ammonium salt solution of Comparative Example 1 just prepared, Figure 1 b is the appearance of the ammonium salt solution of Comparative Example 1 when placed at 4°C for 2 weeks. Figure 1 c is an appearance diagram of the ammonium salt solution of Comparative Example 1 after being placed at 37° C. for 2 weeks;

[0043] Figure 2 The following are pictures obtained by performing a dyeing experiment using the freshly prepared ammonium salt solution of Comparative Example 1 and the ammonium salt solution of Comparative Example 1 after being stored at 37° C. for 2 weeks according to the method of Example 17 in Experimental Example 1; wherein, Figure 2a is a picture after staining with the newly prepared ammonium salt solution of Comparative Example 1 and Alcian blue staining solution, Figure 2 b is a picture after staining with the ammonium salt solution of Comparative Example 1 and Alcian blue staining solution after being placed at 37°C for 2 weeks. Figure 2 c is a picture after counterstaining with the newly prepared ammonium salt solution of Comparative Example 1 and Nuclear Fast Red staining solution, Figure 2 d is a picture after counterstaining with the ammonium salt solution of Comparative Example 1 and Nuclear Fast Red staining solution after being placed at 37°C for 2 weeks;

[0044] Figure 3 This is a picture with a magnification of 4 obtained by performing a staining experiment using the freshly prepared ammonium salt solution of Comparative Example 1 and the ammonium salt solution of Comparative Example 1 after being stored at 37° C. for 2 weeks according to the method of Example 17 in Experimental Example 1; wherein, Figure 3 a is a picture at a magnification of 4 after counterstaining with the newly prepared ammonium salt solution of Comparative Example 1 and Nuclear Fast Red Staining Solution, Figure 3 b is a picture at a magnification of 4 after counterstaining with the ammonium salt solution of Comparative Example 1 and Nuclear Fast Red staining solution after being placed at 37°C for 2 weeks;

[0045] Figure 4 The storage stability test results of the ammonium salt solutions of Examples 1-7 and Comparative Example 1 in Experimental Example 2 are shown in FIG. Figure 4 a is an appearance diagram of the ammonium salt solutions of Examples 1-7 and Comparative Example 1 at 0 day of storage; Figure 4 b is an appearance diagram of the ammonium salt solutions of Examples 1-7 and Comparative Example 1 after storage for 14 days; Figure 4 c is an appearance diagram of the ammonium salt solutions of Examples 1-7 and Comparative Example 1 when stored for 90 days; Figure 4 d is an appearance diagram of the ammonium salt solutions of Examples 1-7 and Comparative Example 1 after storage for 180 days; Figure 4 e is an appearance diagram of the ammonium salt solutions of Examples 1-7 and Comparative Example 1 when stored for 365 days; Figure 4 In a-4e, the samples from left to right are the ammonium salt solutions of Comparative Example 1 and Examples 1-7, respectively;

[0046] Figure 5 The following are the appearance diagrams of the ammonium salt solutions of Comparative Example 1 and Examples 1-7 in Experimental Example 3 when placed at 37° C. for 0, 3, and 7 days; wherein, Figure 5 a is the appearance of the ammonium salt solutions of Comparative Example 1 and Examples 1-7 after being placed at 37° C. for 0 day; Figure 5 b is the appearance of the ammonium salt solutions of Comparative Example 1 and Examples 1-7 when placed at 37° C. for 3 days; Figure 5 c is the appearance of the ammonium salt solutions of Comparative Example 1 and Examples 1-7 after being placed at 37° C. for 7 days; Figure 5In a-5c, the samples from left to right are the ammonium salt solutions of Comparative Example 1 and Examples 1-7, respectively;

[0047] Figure 6 The following are the appearance diagrams of the ammonium salt solutions of Comparative Example 2 and Examples 8-14 in Experimental Example 3 after being placed at 37° C. for 0, 3, and 7 days; wherein, Figure 6 a is the appearance of the ammonium salt solutions of Comparative Example 2 and Examples 8-14 after being placed at 37° C. for 0 day; Figure 6 b is the appearance of the ammonium salt solutions of Comparative Example 2 and Examples 8-14 when placed at 37° C. for 3 days; Figure 6 c is the appearance of the ammonium salt solutions of Comparative Example 2 and Examples 8-14 after being placed at 37° C. for 7 days; Figure 6 In a-6c, the samples from left to right are the ammonium salt solutions of Comparative Example 2 and Examples 8-14, respectively;

[0048] Figure 7 This is the appearance of the ammonium salt solutions of Comparative Example 2 and Examples 8-14 in Experimental Example 4 after being placed at 4° C. for 30 days;

[0049] Figure 8 The following are appearance diagrams of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 3-10 in Experimental Example 5 after being placed at 37° C. for 0 days, 3 days, and 7 days; wherein, Figure 8 a is the appearance of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 3-10 when placed at 37° C. for 0 day; Figure 8 b is the appearance of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 3-10 when placed at 37° C. for 3 days; Figure 8 c is the appearance of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 3-10 when placed at 37° C. for 7 days; Figure 8 In a-8c, the samples from left to right are the ammonium salt solutions of Comparative Example 1 and Comparative Examples 3-10 respectively;

[0050] Figure 9 The following are appearance diagrams of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 11-19 in Experimental Example 5 after being placed at 37° C. for 0 days, 3 days, and 7 days; wherein, Figure 9 a is the appearance of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 11-19 when placed at 37° C. for 0 day; Figure 9 b is the appearance of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 11-19 when placed at 37° C. for 3 days; Figure 9 c is the appearance of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 11-19 when placed at 37° C. for 7 days; Figure 9 In a-9c, the samples from left to right are the ammonium salt solutions of Comparative Example 1 and Comparative Examples 11-19 respectively;

[0051] Figure 10The following are appearance diagrams of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 20-24 in Experimental Example 5 after being placed at 37° C. for 0 days, 3 days, and 7 days; wherein, Figure 10 a is the appearance of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 20-24 when placed at 37° C. for 0 day; Figure 10 b is the appearance of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 20-24 when placed at 37° C. for 3 days; Figure 10 c is the appearance of the ammonium salt solutions of Comparative Example 1 and Comparative Examples 20-24 when placed at 37° C. for 7 days; Figure 10 In a-10c, the samples from left to right are the ammonium salt solutions of Comparative Example 1 and Comparative Examples 20-24 respectively;

[0052] Figure 11 The results of the dyeing experiment in Experimental Example 7 using the freshly prepared ammonium salt solution of Example 1 and the ammonium salt solutions of Examples 1-7 and Comparative Example 1 after being placed in an environment of 2 to 7° C. for 90 days according to the method of Example 17 are shown; wherein, Figure 11 a is a picture obtained by dyeing with the ammonium salt solution prepared in Example 1; Figure 11 b is a picture obtained by dyeing with the ammonium salt solution of Comparative Example 1 after being placed in an environment of 2-7°C for 90 days; Figure 11 c is a picture obtained by staining with the ammonium salt solution of Example 1 after being placed in an environment of 2-7°C for 90 days; Figure 11 d is a picture obtained by staining with the ammonium salt solution of Example 2 after being placed in an environment of 2-7°C for 90 days; Figure 11 e is a picture obtained by staining with the ammonium salt solution of Example 3 after being placed in an environment of 2-7°C for 90 days; Figure 11 f is a picture obtained by staining with the ammonium salt solution of Example 4 after being placed in an environment of 2-7°C for 90 days; Figure 11 g is a picture obtained by staining with the ammonium salt solution of Example 5 after being placed in an environment of 2-7°C for 90 days; Figure 11 h is a picture obtained by staining with the ammonium salt solution of Example 6 after being placed in an environment of 2-7°C for 90 days; Figure 11 i is a picture obtained by staining with the ammonium salt solution of Example 7 after being placed in an environment of 2-7°C for 90 days;

[0053] Figure 12 This is a picture obtained at a magnification of 4 obtained by performing a staining experiment using the ammonium salt solutions of Examples 1-4 placed in an environment of 2-7°C for 90 days according to the method of Example 17 in Experimental Example 7; wherein, Figure 12 a is a picture at a magnification of 4 obtained by performing a staining experiment using the ammonium salt solution of Example 1 after being placed in an environment of 2-7°C for 90 days according to the method of Example 17; Figure 12b is a picture at a magnification of 4 obtained by performing a staining experiment using the ammonium salt solution of Example 2 after being placed in an environment of 2-7° C. for 90 days according to the method of Example 17; Figure 12 c is a picture at a magnification of 4 obtained by performing a staining experiment using the ammonium salt solution of Example 3 after being placed in an environment of 2-7° C. for 90 days according to the method of Example 17; Figure 12 d is a picture at a magnification of 4 obtained by performing a staining experiment using the ammonium salt solution of Example 4 after being placed in an environment of 2-7° C. for 90 days according to the method of Example 17;

[0054] Figure 13 This is a picture obtained at a magnification of 4 obtained by staining the ammonium salt solutions of Examples 5-7 in Experimental Example 7 after being placed in an environment of 2-7°C for 90 days according to the method of Example 17; wherein, Figure 13 a is a picture at a magnification of 4 obtained by performing a staining experiment using the ammonium salt solution of Example 5 after being placed in an environment of 2-7°C for 90 days according to the method of Example 17; Figure 13 b is a picture at a magnification of 4 obtained by performing a staining experiment using the ammonium salt solution of Example 6 after being placed in an environment of 2-7° C. for 90 days according to the method of Example 17; Figure 13 c is a picture at a magnification of 4 obtained by performing a staining experiment using the ammonium salt solution of Example 7 after being placed in an environment of 2-7° C. for 90 days according to the method of Example 17;

[0055] Figure 14 The diagram shows the results of staining the sulfated acidic mucus and sialic acid mucus using the ammonium salt solutions of Example 12 and Comparative Example 2 after being placed at 37° C. for different times (0 to 14 days) in Experimental Example 8; wherein, Figure 14 a is a diagram showing the results of differential staining of sulfated acidic mucus and sialic acid mucus using the ammonium salt solution of Comparative Example 2 after being placed at 37° C. for different times (0 to 14 days); Figure 14 b is a diagram showing the results of differential staining of sulfated acidic mucus and sialic acid mucus using the ammonium salt solution of Example 12 after being placed at 37° C. for different periods of time (0 to 14 days);

[0056] Figure 15 This is a picture of the differential staining results of sulfated acidic mucus and sialic acid mucus using the ammonium salt solutions of Example 12 and Comparative Example 2 after being placed at 37°C for different times (0 to 14 days) in Experimental Example 8, at a magnification of 4; wherein, Figure 15 a is a picture of the differential staining results of sulfated acidic mucus and sialic acid mucus using the ammonium salt solution of Comparative Example 2 placed at 37° C. for 0 day, at a magnification of 4; Figure 15b is a picture of the differential staining results of sulfated acidic mucus and sialic acid mucus using the ammonium salt solution of Comparative Example 2 placed at 37° C. for 6 days, at a magnification of 4; Figure 15 c is a picture of the differential staining results of sulfated acidic mucus and sialic acid mucus using the ammonium salt solution of Example 12 placed at 37° C. for 0 day, at a magnification of 4; Figure 15 d is a picture at a magnification of 4 showing the differential staining results of sulfated acidic mucus and sialic acid mucus using the ammonium salt solution of Example 12 that was placed at 37° C. for 8 days;

[0057] Figure 16 This is a picture obtained when the ammonium salt solution of Comparative Example 20 is used for dyeing in Experimental Example 9. DETAILED DESCRIPTION

[0058] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0059] 1. Specific embodiments of the ammonium salt solution of the present invention are as follows:

[0060] The ammonium salt solutions of Examples 1-7 were prepared from N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent, and water. The mass ratio of N,N-dimethyl-m-phenylenediamine dihydrochloride to water was 0.72:50. The metal ion chelating agent was disodium ethylenediaminetetraacetate, and the concentration of the metal ion chelating agent was x mmol / L. The amounts of the metal ion chelating agent used in the ammonium salt solutions of Examples 1-7 are shown in Table 1.

[0061] Table 1 Amount of metal ion chelating agent in the ammonium salt solution of Examples 1-7

[0062] Ammonium salt solution x Example 1 0.001 Example 2 0.005 Example 3 0.01 Example 4 0.05 Example 5 0.1 Example 6 0.5 Example 7 1

[0063] The ammonium salt solutions of Examples 8-14 were prepared from N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent, and water. The mass ratio of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, and water was 0.12:0.72:50. The metal ion chelating agent was disodium ethylenediaminetetraacetate, and the concentration of the metal ion chelating agent was μ mmol / L. The amounts of the metal ion chelating agents used in the ammonium salt solutions of Examples 8-14 are shown in Table 2.

[0064] Table 2 Amount of metal ion chelating agent in the ammonium salt solution of Examples 8-14

[0065] Ammonium salt solution y Example 8 1 Example 9 0.5 Example 10 0.1 Example 11 0.05 Example 12 0.01 Example 13 0.005 Example 14 0.001

[0066] Comparative Example 1

[0067] The only difference between the ammonium salt solution of this comparative example and the ammonium salt solution of Example 1 is that the concentration of the metal ion chelating agent in the ammonium salt solution of this comparative example is 0 mmol / L.

[0068] Comparative Example 2

[0069] The only difference between the ammonium salt solution of this comparative example and the ammonium salt solution of Example 8 is that the concentration of the metal ion chelating agent in the ammonium salt solution of this comparative example is 0 mmol / L.

[0070] The ammonium salt solution of Comparative Example 3-24 is prepared from N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the mass ratio of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride and water is 0.12:0.72:50, and the mass fraction of the metal ion chelating agent is z%. The type and amount of the metal ion chelating agent in the ammonium salt solution of Comparative Example 3-24 are shown in Table 3.

[0071] Table 3 Type and dosage of metal ion chelating agent in ammonium salt solution of Comparative Examples 3-24

[0072]

[0073]

[0074] 2. Specific examples of the ferrous diamine-alecian blue staining solution of the present invention are as follows:

[0075] Example 15

[0076] The ferric diamine-alcian blue staining solution of this embodiment includes ferric diamine solution, alcian blue staining solution and nuclear fast red staining solution;

[0077] Wherein, the high iron diamine solution includes N,N-dimethyl-p-phenylenediamine dihydrochloride solution, ferric chloride solution and N,N-dimethyl-m-phenylenediamine dihydrochloride solution; N,N-dimethyl-p-phenylenediamine dihydrochloride solution is prepared by dissolving 0.12 parts by mass of N,N-dimethyl-p-phenylenediamine dihydrochloride in 50 parts by mass of water; the ferric chloride solution is prepared by dissolving 5.4 parts by mass of ferric chloride in 100 parts by mass of water; the N,N-dimethyl-m-phenylenediamine dihydrochloride solution is the ammonium salt solution of Example 1;

[0078] The Alcian blue staining solution is prepared by dissolving Alcian blue in water and then adjusting the pH. The mass ratio of Alcian blue to water is 1:100. The pH adjuster is acetic acid. The pH of the Alcian blue staining solution is 2.5.

[0079] The nuclear fast red staining solution is prepared by a method comprising the following steps: first dissolving aluminum sulfate in purified water, heating to completely dissolve the aluminum sulfate, adding nuclear fast red after the aluminum sulfate is completely dissolved, stirring to fully dissolve the nuclear fast red, and filtering to obtain the solution; the mass ratio of aluminum sulfate, nuclear fast red, and water is 5:0.1:100.

[0080] In other embodiments, the N,N-dimethyl-p-phenylenediamine dihydrochloride solution in the ferric diamine-alecian blue staining solution of Example 15 may be replaced with any ammonium salt solution in Examples 2-7.

[0081] Example 16

[0082] The ferric diamine-alcian blue staining solution of this embodiment includes ferric diamine solution, alcian blue staining solution and nuclear fast red staining solution;

[0083] The high-iron diamine solution includes an ammonium salt solution and a ferric chloride solution, the ammonium salt solution is the ammonium salt solution of Example 8; the ferric chloride solution is prepared by dissolving 3.6 parts by mass of ferric chloride in 100 parts by mass of water;

[0084] The Alcian blue staining solution and the nuclear fast red staining solution are the same as the ferric chloride solution, Alcian blue staining solution and the nuclear fast red staining solution used in Example 15.

[0085] In other embodiments, the ammonium salt solution in the ferric diamine-alecian blue staining solution of Example 16 may be replaced with any of the ammonium salt solutions in Examples 9-14.

[0086] 3. Specific examples of the use of the ferric diamine-alecian blue staining solution of the present invention in the differential staining of sulfated acidic mucus and sialic acid mucus are as follows:

[0087] Example 17

[0088] The use of the ferric diamine-alecian blue staining solution of this embodiment in the differential staining of sulfated acidic mucus and sialic acid mucus specifically includes the following steps:

[0089] (1) Slice, dewax, and hydrate the intestinal tissue;

[0090] (2) Mix equal volumes of N,N-dimethyl-p-phenylenediamine dihydrochloride solution, N,N-dimethyl-m-phenylenediamine dihydrochloride solution, and ferric chloride solution in the ferric diamine-alecian blue staining solution of Example 15 (volume ratio of 1:1:1) and add dropwise to the intestinal tissue sections, incubate at room temperature for 18 h, and then rinse with distilled water;

[0091] (3) The washed intestinal tissue was then stained with the Alcian blue staining solution in Example 15 for 15 minutes, and then rinsed with distilled water;

[0092] (4) After rinsing, the intestinal tissue was counterstained with the nuclear fast red staining solution in the high iron diamine-alecian blue staining solution of Example 15 for 3 minutes, and finally routinely dehydrated, transparentized, and sealed with neutral gum for microscopic examination.

[0093] In other embodiments, equal volumes of N,N-dimethyl-p-phenylenediamine dihydrochloride solution, N,N-dimethyl-m-phenylenediamine dihydrochloride solution and ferric chloride solution in the ferric diamine-alcian blue staining solution of Example 15 were mixed (volume ratio of 1:1:1) and then dropped onto the intestinal tissue. The incubation time at room temperature was 20 and 24 hours, the Alcian blue staining solution was used to stain the flushed intestinal tissue for 15 and 20 minutes, and the nuclear fast red staining solution was used to re-stain the intestinal tissue for 4 and 5 minutes.

[0094] Example 18

[0095] The use of the ferric diamine-alecian blue staining solution of this embodiment in the differential staining of sulfated acidic mucus and sialic acid mucus specifically includes the following steps:

[0096] (1) Slice, dewax, and hydrate the intestinal tissue;

[0097] (2) The ammonium salt solution and the ferric chloride solution in the ferric diamine-alecian blue staining solution of Example 16 were mixed in equal volumes (volume ratio of 1:1) and then added dropwise to the intestinal tissue sections. The mixture was incubated at room temperature for 18 h, and then rinsed with distilled water.

[0098] (3) The washed intestinal tissue was then stained with the Alcian blue staining solution in Example 16 for 15 minutes, and then rinsed with distilled water;

[0099] (4) After rinsing, the intestinal tissue was counterstained with the nuclear fast red staining solution in the high iron diamine-alecian blue staining solution of Example 16 for 3 minutes, and finally conventionally dehydrated, transparentized, and sealed with neutral gum for microscopic examination.

[0100] Experimental Example 1

[0101] In order to investigate the color change of the ammonium salt solution of Comparative Example 1 after being placed for a certain period of time and the effect of placing for a certain period of time on the dyeing results, the ammonium salt solution of Comparative Example 1 was placed at 4°C and 37°C for 2 weeks respectively. The color change results are shown in FIG. Figure 1 Then, the ammonium salt solution of Comparative Example 1 prepared just now and the ammonium salt solution of Comparative Example 1 placed at 37°C for 2 weeks were dyed according to the method of Example 17. The dyeing results are shown in FIG. Figure 2-3 As shown. Among them, Figure 2 a is a picture after staining with the newly prepared ammonium salt solution of Comparative Example 1 and Alcian blue staining solution, Figure 2 b is a picture after staining with the ammonium salt solution of Comparative Example 1 and Alcian blue staining solution after being placed at 37°C for 2 weeks. Figure 2 c is a picture after counterstaining with the newly prepared ammonium salt solution of Comparative Example 1 and Nuclear Fast Red staining solution, Figure 2 d is a picture after counterstaining with the ammonium salt solution of Comparative Example 1 and Nuclear Fast Red staining solution after being placed at 37°C for 2 weeks. Figure 3 a is a picture at a magnification of 4 after counterstaining with the newly prepared ammonium salt solution of Comparative Example 1 and Nuclear Fast Red Staining Solution, Figure 3 b is a picture at a magnification of 4 after counterstaining with the ammonium salt solution of Comparative Example 1 and Nuclear Fast Red staining solution after being placed at 37° C. for 2 weeks.

[0102] Depend on Figure 1 It can be seen that the ammonium salt solution of Comparative Example 1 will be oxidized even when stored at 4°C, resulting in a color change, and the longer the storage time, the darker the color. After being stored at 37°C for 2 weeks, its color directly turns black.

[0103] Depend on Figure 2-3 It can be seen that after the ammonium salt solution of Comparative Example 1 was placed at 37°C for 2 weeks, the staining results when used for staining were significantly different from those of the freshly prepared ammonium salt solution. The ratio of sulfated acidic mucus substances to sialic acid mucus substances explained by the staining results underwent a qualitative change, thus affecting the pathological diagnosis.

[0104] Experimental Example 2

[0105] In order to evaluate the storage stability of the ammonium salt solutions of Examples 1-7 and Comparative Example 1, the ammonium salt solutions of Examples 1-7 and Comparative Example 1 were placed in an environment of 2-7°C for 0, 14, 90, 180, and 365 days, and the colors of the ammonium salt solutions at different placement times were observed and recorded. The experimental results are shown in FIG. Figure 4 As shown. Among them, Figure 4 a-4d are the appearance diagrams of the N,N-dimethyl-m-phenylenediamine dihydrochloride solutions of Examples 1-7 and Comparative Example 1 in Experimental Example 1 after storage for 0, 14, 90, 180, and 365 days, respectively. Figure 4 In a-4e, the samples from left to right are the ammonium salt solutions of Comparative Example 1 and Examples 1-7, respectively.

[0106] The results showed that the freshly prepared ammonium salt solutions were all transparent and colorless liquids. After 14 days, the ammonium salt solutions of Examples 3-7 and Comparative Example 1 all showed discoloration, with the discoloration of the ammonium salt solution of Comparative Example 1 being more severe. After 90, 180, and 365 days, only the ammonium salt solution of Example 1 had the lightest color, while the ammonium salt solutions of Example 7 and Comparative Example 1 had the darkest colors. These results demonstrate that disodium EDTA can significantly slow the discoloration of the dyeing solution, and that the concentration of disodium EDTA in the N,N-dimethyl-m-phenylenediamine dihydrochloride solution needs to be controlled within a certain range to achieve a good protective effect.

[0107] Experimental Example 3

[0108] It's long been known that temperature significantly affects the rate of chemical reactions. Vant Hoff, based on a rough rule derived from experiments, states that every 10°C increase in temperature increases the reaction rate by approximately 2-4 times. In this experiment, 37°C was used to simulate storage conditions at 2-8°C. This means that one week of storage at 37°C is equivalent to 4-16 weeks at 4°C.

[0109] The ammonium salt solutions of Comparative Example 1-2 and Example 1-14 were placed at 37°C for 0, 3, and 7 days, and the colors of the ammonium salt solutions at different times were observed and recorded. The colors of the ammonium salt solutions of Comparative Example 1 and Example 1-7 at different times were shown in the figure. Figure 5 The color results of the ammonium salt solutions of Comparative Example 2 and Examples 8-14 at different times are shown in FIG. Figure 6 shown.

[0110] The results show that compared with Comparative Examples 1-2, the color of the ammonium salt solution of Example 1-14 is lighter after being placed at 37°C for a certain period of time, indicating that disodium ethylenediaminetetraacetic acid can slow down the discoloration of N,N-dimethyl-m-phenylenediamine dihydrochloride and / or N,N-dimethyl-p-phenylenediamine dihydrochloride and improve its stability.

[0111] Experimental Example 4

[0112] In order to further investigate the stabilizing effect of disodium ethylenediaminetetraacetate on the solution containing N,N-dimethyl-m-phenylenediamine dihydrochloride and N,N-dimethyl-p-phenylenediamine dihydrochloride, the ammonium salt solutions of Comparative Example 2 and Examples 8-14 were placed at 4°C for 30 days, and then the color of each ammonium salt solution was observed and recorded. The results are as follows: Figure 7 The results show that disodium EDTA has a good stabilizing effect on solutions containing N,N-dimethyl-m-phenylenediamine dihydrochloride and N,N-dimethyl-p-phenylenediamine dihydrochloride, and can slow down their discoloration.

[0113] Experimental Example 5

[0114] In order to investigate the stabilizing effect of other metal ion chelating agents on solutions containing N,N-dimethyl-m-phenylenediamine dihydrochloride and N,N-dimethyl-p-phenylenediamine dihydrochloride, the ammonium salt solutions of Comparative Example 1 and Comparative Example 3-24 were placed at 37°C for 0 days, 3 days, and 7 days, and the colors of the ammonium salt solutions at different times were observed and recorded. The appearance of the ammonium salt solutions of Comparative Example 1 and Comparative Example 3-10 at 37°C for 0 days, 3 days, and 7 days are shown in the following pictures. Figure 8 As shown; Comparative Example 1 and Comparative Example 11-19 ammonium salt solution at 37 ° C for 0d, 3d, 7d when the appearance of the picture as shown Figure 9 As shown; Comparative Example 1 and Comparative Example 20-24 of the ammonium salt solution at 37 ° C for 0d, 3d, 7d when the appearance of the picture as shown Figure 10 The results showed that phytic acid, sodium pyrophosphate, tartaric acid, citric acid, sodium metasilicate, and glycerol had no stabilizing effect on solutions containing N,N-dimethyl-m-phenylenediamine dihydrochloride and N,N-dimethyl-p-phenylenediamine dihydrochloride. Diethylenetriamine penta (methylenephosphonic acid) had a weak protective effect on solutions containing N,N-dimethyl-m-phenylenediamine dihydrochloride and N,N-dimethyl-p-phenylenediamine dihydrochloride.

[0115] Experimental Example 6

[0116] In order to further quantify the color change of the ammonium salt solution of embodiment 1-7 and embodiment 8-14, OD value detection is carried out by ultraviolet spectrophotometry (Shanghai Yidian Analytical Instrument Co., Ltd., model L5S) to monitor the discoloration degree of the ammonium salt solution. The ammonium salt solution of embodiment 12 and comparative example 2 placed at 37 ℃ for 14d is scanned with visible light wavelength (350nm-750nm), and it is found that the maximum absorption wavelength is 550nm. Then the ammonium salt solution of embodiment 12 and comparative example 2 placed at 37 ℃ for different times is detected, and the OD value is calculated again. The result is as shown in Table 4.

[0117] Table 4 OD values ​​of ammonium salt solutions of Example 12 and Comparative Example 2 when placed for different times

[0118]

[0119] The results show that the OD values ​​of the ammonium salt solutions of Example 12 and Comparative Example 2 begin to differ significantly on the second day of storage, indicating that the ammonium salt solution of Example 12 has better stability and less discoloration.

[0120] Experimental Example 7

[0121] In order to evaluate the effect of the ammonium salt solutions of Examples 1-7 and Comparative Example 1 on the identification and staining of sulfated acidic mucus and sialic acid mucus when they were just prepared and when they were placed in an environment of 2-7°C for 90 days, the ammonium salt solutions of Example 1 just prepared and the ammonium salt solutions of Examples 1-7 and Comparative Example 1 placed in an environment of 2-7°C for 90 days were dyed according to the method of Example 17. The dyeing results are shown in FIG. Figure 11-13 The results show that the best dyeing effect is achieved when the freshly prepared ammonium salt solution is used for dyeing. When the ammonium salt solution of Comparative Example 1 is placed in an environment of 2-7°C for 90 days, the dyeing intensity is weakened when the ammonium salt solution of Comparative Example 1 is used for dyeing, while the dyeing effect is not significantly weakened when the ammonium salt solutions of Examples 1-7 are used for dyeing, and the background is clean.

[0122] Experimental Example 8

[0123] In order to evaluate the effect of the ammonium salt solutions of Example 12 and Comparative Example 2 on the dyeing results after being placed at 37°C for different times, the ammonium salt solutions placed for different times (0 to 14 days) were dyed according to the method of Example 18. The dyeing results are shown in FIG. Figure 14-15 The results showed that after 6 and 8 days at 37°C, the intestinal paraffin sections showed obvious differences in staining effect. The proportion of brown-purple to brown-black acidic mucin began to increase, and the proportion of blue sialic acid mucin began to decrease. Therefore, the ammonium salt solution of Example 12 has better stability and staining effect.

[0124] Experimental Example 9

[0125] In order to evaluate the effect of the ammonium salt solution of Comparative Examples 20-21 on the dyeing results, the ammonium salt solution of Comparative Examples 20-21 just prepared was subjected to a dyeing experiment according to the method of Example 18. The dyeing results when the ammonium salt solution of Comparative Example 20 was used for dyeing are shown in FIG. Figure 16 The results show that when the ammonium salt solutions of Comparative Examples 20-21 are used for dyeing, the dyeing results all show only blue, indicating that the ammonium salt solutions of Comparative Examples 20-21 can only identify sialic acid mucus substances when used for dyeing, but cannot identify sulfated acidic mucus substances. Therefore, the ammonium salt solution containing diethylenetriamine penta (methylene phosphonic acid) is not suitable for the differential dyeing of sulfated acidic mucus substances and sialic acid mucus substances.

Claims

1. An ammonium salt solution, characterized in that The ammonium salt solution is made of N, N- dimethyl - m - phenylenediamine dihydrochloride, a metal ion chelating agent and water; or the ammonium salt solution is made of N, N- dimethyl - p - phenylenediamine dihydrochloride, N, N- dimethyl - m - phenylenediamine dihydrochloride, a metal ion chelating agent and water; the metal ion chelating agent is disodium ethylenediaminetetraacetate; When the ammonium salt solution is prepared from N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the mass ratio of the N,N-dimethyl-m-phenylenediamine dihydrochloride to water is (0.12-0.72):50, and the amount of the metal ion chelating agent used for every 0.72 g of N,N-dimethyl-m-phenylenediamine dihydrochloride is 5×10 -8 ~5×10 -5 mol; When the ammonium salt solution is prepared from N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent, and water, the mass ratio of the N,N-dimethyl-m-phenylenediamine dihydrochloride, N,N-dimethyl-p-phenylenediamine dihydrochloride, and water is (0.12-0.72):(0.02-0.12):50, and the amount of the metal ion chelating agent used per 0.72 g of N,N-dimethyl-m-phenylenediamine dihydrochloride is 5×10 -8 ~5×10 -5 mol.

2. The ammonium salt solution according to claim 1, wherein When the ammonium salt solution is made of N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the amount of the metal ion chelating agent used for each 0.72 g of N,N-dimethyl-m-phenylenediamine dihydrochloride is 5×10 -8 mol; When the ammonium salt solution is made of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the amount of the metal ion chelating agent used for each 0.72 g of N,N-dimethyl-m-phenylenediamine dihydrochloride is 5×10 -8 ~5×10 -6 mol.

3. A high iron diamine-alecian blue staining solution, characterized in that, The invention comprises a ferric diamine solution and an Alcian blue staining solution, wherein the ferric diamine solution comprises a ferric chloride solution and an ammonium salt solution according to any one of claims 1 to 2; when the ammonium salt solution is made of N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the ferric diamine solution further comprises an N,N-dimethyl-p-phenylenediamine dihydrochloride solution.

4. The ferrous diamine-alecian blue staining solution according to claim 3, wherein The ferric diamine-alecian blue staining solution also includes nuclear fast red staining solution.

5. The ferrous diamine-alecian blue staining solution according to claim 3, wherein The N,N-dimethyl-p-phenylenediamine dihydrochloride solution is prepared by dissolving 0.02 to 0.12 parts by mass of N,N-dimethyl-p-phenylenediamine dihydrochloride in 50 parts by mass of water; The ferric chloride solution is prepared by dissolving 3.6 to 5.4 parts by mass of ferric chloride in 100 parts by mass of water; The Alcian blue staining solution is made of Alcian blue, a pH regulator and water; the mass ratio of the Alcian blue to water is 1:100; the pH of the Alcian blue staining solution is 2.5; and the pH regulator is acetic acid.

6. Use of the ferric diamine-alecian blue staining solution according to any one of claims 3 to 5 in differential staining of sulfated acidic mucus substances and sialic acid mucus substances.

7. The use according to claim 6, characterized in that The ferric diamine-alecian blue staining solution includes a nuclear fast red staining solution, and the application includes the following steps: (1) When the ammonium salt solution in the ferric diamine-alecian blue staining solution is made of N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the ammonium salt solution in the ferric diamine-alecian blue staining solution, the N,N-dimethyl-p-phenylenediamine dihydrochloride solution and the ferric chloride solution are mixed and applied to the surface of the object to be tested, followed by incubation. After the incubation is completed, the object is rinsed with water; When the ammonium salt solution in the ferric diamine-alecian blue staining solution is made of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelator, and water, the ammonium salt solution in the ferric diamine-alecian blue staining solution and the ferric chloride solution are mixed and applied to the surface of the object to be tested, followed by incubation, and after the incubation is completed, the object is rinsed with water; (2) staining the sample obtained in step (1) with the alecian blue staining solution in the ferrous diamine-alecian blue staining solution, and rinsing with water after staining; (3) using the nuclear fast red staining solution in the ferric diamine-alecian blue staining solution to counterstain the stained sample obtained in step (2); (4) Observe the color of the test substance after re-staining to complete the identification of sulfated acidic mucus substances and sialic acid mucus substances.

8. The use according to claim 7, characterized in that When the ammonium salt solution in the ferric diamine-alecian blue staining solution is made of N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the volume ratio of the ammonium salt solution, N,N-dimethyl-p-phenylenediamine dihydrochloride solution and the ferric chloride solution is 1:1:1; when the ammonium salt solution in the ferric diamine-alecian blue staining solution is made of N,N-dimethyl-p-phenylenediamine dihydrochloride, N,N-dimethyl-m-phenylenediamine dihydrochloride, a metal ion chelating agent and water, the volume ratio of the ammonium salt solution to the ferric chloride solution is 1:1.

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