CN108342359B

CN108342359B - Preparation method of digestive juice

Info

Publication number: CN108342359B
Application number: CN201810467929.3A
Authority: CN
Inventors: 王诚; 陆晓青; 余美文; 侯传玲; 姚育英; 孙爱静
Original assignee: Shaoxing Peoples Hospital
Current assignee: Shaoxing Peoples Hospital
Priority date: 2018-05-16
Filing date: 2018-05-16
Publication date: 2021-04-13
Anticipated expiration: 2038-05-16
Also published as: CN108342359A

Abstract

The application relates to a preparation method of digestive juice, and belongs to the technical field of sample preparation for testing. Weighing EDTA powder, dissolving the EDTA powder in PBS, adjusting the pH value of the solution to be proper during preparation, and stirring uniformly to prepare EDTA mother liquor; respectively weighing trypsin and proteinase K, dissolving the trypsin and proteinase K in a PBS solution, then dripping the solution into EDTA mother solution in proportion, and supplementing and dripping PBS according to needs, wherein the formed digestive juice comprises the following components in concentration: proteinase K0.1-2 mg/ml, trypsin 1-100mg/ml and EDTA0.1-2 mg/ml. The application is applied to the treatment of tumor lymph node tissue specimens, particularly tumor lymph node radical treatment specimens treated by combining a stationary liquid and acid and alkali, the hardness difference of fat tissues and lymph nodes is increased, the lymph node materials are conveniently taken, and the lymph node detection number and the accurate lymph node metastasis positive rate are effectively improved.

Description

Preparation method of digestive juice

Technical Field

The application relates to a preparation method of digestive juice, and belongs to the technical field of sample preparation for testing.

Background

The existing tumor lymph node cleaning in vitro tissue specimen is usually soaked in formalin fixing solution, which is mainly because formaldehyde can well preserve the histomorpholological structural characteristics for a long time and has good economic utility. Although the specimen processing method can fix the tissue form well, the specimen containing adipose tissue is also hardened, which greatly increases the difficulty in obtaining the material in the pathology department, especially in obtaining the lymph node. In the tumor lymph node cleaning isolated tissue specimen, thorough lymph node material taking is crucial to the staging of tumor diagnosis, the establishment of a treatment scheme and the judgment of prognosis, and the conventional fixing solution and the existing specimen fixing method cannot meet the requirements of improving the material taking speed and the detection rate of the lymph node while quickly fixing, so that the method for searching the lymph node by a pathologist in a long-term isolated adipose tissue by touching a knife has the defects of insufficient material taking, long operation time and the like, and finally the treatment scheme, prognosis and outcome of a patient are influenced. In order to solve the problems in the existing technology for processing the tumor lymph node cleaning in vitro tissue specimen, a method for fully exposing and fixing lymph nodes and keeping the tissue structure and the cell morphology of the lymph node complete while softening or dissolving fat is urgently needed in clinical practical work.

The present application was made based on this.

Disclosure of Invention

Aiming at the defects of the existing lymph node detection technology, the application provides a brand-new preparation method of the fixed fat digestive juice, fat tissue and lymph node hardness are increased while fat is digested and liquefied, and lymph node material taking is facilitated.

In order to achieve the purpose, the technical scheme adopted by the application is as follows:

a preparation method of digestive juice comprises weighing EDTA powder, dissolving in PBS, adjusting pH of the solution to appropriate value during preparation, stirring well, and preparing EDTA mother liquor; respectively weighing trypsin and proteinase K, dissolving the trypsin and proteinase K in a PBS solution, then dripping the solution into EDTA mother solution in proportion, and supplementing and dripping PBS according to needs, wherein the formed digestive juice comprises the following components in concentration: proteinase K0.1-2 mg/ml, trypsin 1-100mg/ml and EDTA0.1-2 mg/ml.

Further, as preferable:

the digestive juices with the above characteristics can also be dissolved in two buffers:

one is PBS buffer solution, pH7.4, which mainly serves to buffer and maintain the pH value and salt ion concentration of the reaction system. Optionally, 1-10mM CaCl may be added₂Divalent cations required for stabilization of the enzyme activity.

The other is pH 7.550mM Tris-HCl buffer solution, and 1-10mM CaCl can be added₂The preparation method is that 6 g Tris-Base is added with water to 900ml, is adjusted to pH7.5 by HCl, and can also be added with CaCl₂To a final concentration of 10 mM. Tris is mainly used for buffering and maintaining the pH value and the salt ion concentration of a reaction system, CaCl₂Divalent cations required for stabilization of the enzyme activity. In the digestive juice, the concentrations of the components are respectively as follows: proteinase K0.5 mg/ml, trypsin 25mg/ml, EDTA1 mg/ml.

The method is applied to the fixation treatment of the tumor lymph node in vitro specimen, and has the advantages that:

(1) the digestive juice provided by the application is a mixed solution with the functions of digesting and liquefying fat and the like, is used for fixing a tumor lymph node radical treatment specimen by 'soft fat fixing solution' and treating with acid and alkali in sequence, can enter fat cells to play a role of digesting fat, can soften and liquefy the specimen fixedly treated by 'soft fat fixing solution', and has no influence or slight influence on lymph nodes because of the fixed protection of a surface compact connective tissue envelope.

(2) After the tissue specimen is treated by the digestive juice, the hardness difference between the adipose tissue and the lymph nodes is increased, the lymph nodes can be conveniently obtained, the working efficiency and the working quality are improved, the anatomical material taking time of doctors in the pathology department is also reduced, and the possibility is provided for the outdoor quality control of the later pathology department.

(4) The application has the advantages that the digestion of the digestive juice on lymph nodes and various tissue specimens is remarkable, the detection number of the lymph nodes is increased, the positive rate of lymph node metastasis is accurate, and the commercial effect is particularly prominent in clinical wide popularization and application.

Drawings

FIG. 1 is a diagram showing the state of fat after the treatment with the fixative in example 3;

FIG. 2 is a graph of the fat state after HCl/PBS treatment in example 3;

FIG. 3 is a graph of the fat state after NaOH/PBS treatment in example 3;

FIG. 4 is a diagram showing the state of fat after the treatment of the digestive juice in example 3;

FIG. 5 is a diagram showing the state of fat after different indwelling treatments in example 3;

FIG. 6 is a graph showing the effect of the treatment method of example 3 on lymph node structure;

FIGS. 7 to 14 are graphs comparing the effects of the treatments under the conditions of example 1 in example 4;

FIGS. 15 to 19 are graphs comparing the effects of the treatments under the conditions of example 2 in example 4;

FIGS. 20 and 21 are graphs showing the effects of the treatment under the conditions of example 3 in example 4;

FIGS. 22, 23 and 24 are graphs showing the effects of the treatment under the conditions of example 4 in example 4;

FIGS. 25, 26 and 27 are graphs showing the effects of the treatments under the conditions of example 5 in example 4;

FIGS. 28 to 35 are graphs comparing the effects of the treatments under the conditions of example 6 in example 4;

FIGS. 36 to 43 are graphs comparing the effects of the treatment under the conditions of example 7 in example 4;

FIGS. 44 to 49 are graphs comparing the effects of the treatment under the conditions of the example 8 in example 4;

FIGS. 50 to 54 are graphs showing the effects of the treatment under the conditions of example 9 in example 4.

Detailed Description

Example 1: composition of digestive juice of different composition

The core action component of the digestive juice provided by the application is digestive enzyme, the main components are proteinase K, trypsin and EDTA, one of PBS or Tris-HCl can be selected as a buffer solution under different implementation environments, and CaCl can be added₂The enzyme activity was stabilized and the specific selection of ingredients was found in Table 1.

TABLE 1 digestive juices with different compositions

Serial number

Proteinase K

Trypsin

EDTA

PBS

Tris-HCl

CaCl₂

1

√

-

2

√

-

3

√

-

√

4

√

-

√

5

√

-

6

√

-

√

7

√

-

√

8

√

The above 8 cases were obtained by testing the digestion solution of the core constituents (i.e. number 1 in table 1) and the complementary constituents separately, and the same samples were tested in parallel for the same components at the same concentrations, which indicated that: the main functions of the core components are to destroy fat cell membranes and increase the permeability of the fat cell membranes. But with supplemental ingredientsThe effect of the buffer solution is not negligible, and the buffer solution is used for buffering and maintaining the pH value and the salt ion concentration of a reaction system; CaCl₂Providing the divalent cation necessary for stabilizing the enzyme activity.

The digestive juice provided by the application has mixed liquid with functions of digesting and liquefying fat and the like, is used for fixing a tumor lymph node radical treatment specimen through 'soft fat fixing liquid' and treating with acid and alkali in sequence, can enter fat cells to play a role in digesting fat, can soften and liquefy the specimen fixed by 'soft fat fixing liquid', and has no influence or slight influence on lymph nodes due to the fixed protection of a surface compact connective tissue envelope. Thereby achieving the purposes of enlarging adipose tissues and improving the hardness difference of lymph nodes, facilitating the material selection of lymph nodes and improving the working efficiency and the working quality. Therefore, the digestive juice having different composition can be selected for the digestion treatment based on different digestion purposes and treatment requirements. The concentration of core components (proteinase K, trypsin and EDTA) and supplementary components in digestive juice is tested, and the results show that in the digestive juice, 0.1-2mg/ml proteinase K, 1-100mg/ml trypsin and 0.1-2mg/ml EDTA can well play the synergistic and cooperative roles, so that ideal balm and lymph node structure can be ensured, and when the concentration is lower than or exceeds the range, the balm is insufficient or the lymph node structure is damaged; when the supplementary components are added, the amounts of the above three core components are kept constant or adjusted within the above ranges, and the addition of the supplementary components is not so large that the fixation solution is easily unbalanced and the softening and dissolving effects are affected, so that the concentrations of the buffers PBS and TRis-HCl are controlled to 1X and 1-10mM (preferably 1mM) of 50mM CaCl2, respectively.

Example 2:

meanwhile, under different working environments, the digestion solution was dissolved in different buffers, and the digestion solution was described below with the solutions in PBS and Tris-HCl as representative examples.

(1) PBS buffer: the pH value of 7.4 mainly plays a role in buffering and maintaining the pH value and the salt ion concentration of the reaction system. In addition, 1-10mM CaCl can be added₂Required for stabilization of enzyme activityA divalent cation.

(2) pH7.550mM Tris-HCl buffer solution, during the preparation process, 1-10mM CaCl can be added₂The preparation method comprises the following steps: 6 g Tris-Base is added with water to 900ml, adjusted to pH7.5 with HCl and optionally CaCl₂To a final concentration of 10 mM. Tris is mainly used for buffering and maintaining the pH value and the salt ion concentration of a reaction system, CaCl₂Divalent cations required for stabilization of the enzyme activity.

Example 3: control of digestive Effect

In this example, six replicates of fresh adipose tissue and lymph nodes excised from a radical gastric carcinoma procedure were treated as follows: (1) the fixing solution is treated for 3 hours at room temperature and consists of the following components in concentration ratio: 100ml/L of formaldehyde, 50g/L of lecithin, 250ml/L of absolute ethyl alcohol, 250ml/L of acetone, 47.5g/L of deoxycholic acid, 250ml/L of methanol, 1 x of PBS solution and 0.1g/L of methylene blue; (2) treating with 0.1N HCl/PBS solution at room temperature for 1 hour; (3) treating with 0.1N NaOH/PBS solution at room temperature for 1 hr; (4) the digestion solution was treated with 25mg of trypsin-1% EDTA/ml + proteinase K, and then treated in a water bath at 37 ℃ for 4 hours, and six samples were subjected to parallel tests using 0.5mg/ml, 0.25mg/ml, 0.1mg/ml, 0.5mg/ml, 0.25mg/ml and 0.1mg/ml of proteinase K, respectively, and the effects of the above-mentioned treatments at the respective stages were observed.

Each tissue specimen after the treatment in step (1) is still hard, as shown in fig. 1; each tissue specimen processed in the step (2) has no obvious change, and six tissue specimens have no difference with each other, which is shown in figure 2; each tissue specimen processed in the step (3) has no obvious change, and six tissue specimens have no difference with each other, which is shown in figure 3; in the tissue specimen processed in the step (4), the adipose tissues of the samples No.1 and No.4 are obviously softened and thinned, the hardness difference between the adipose tissues and the lymph nodes is obvious, the materials are easily identified and taken, and the softening degree of other samples is not obvious, which is shown in fig. 4.

Respectively carrying out retention treatment on the samples treated in the four steps: the samples placed in the fixative solution of the present application still performed well, but due to the insufficient proteinase K concentration of No.2 and No.3, the retention effect of No.1 and No.4 was better than that of No.2 and No.3 at 0.25mg/ml, while the retention of No.5 and No.6 with formalin fixative was less effective, as shown in FIG. 5.

The lymph node tissue obtained by the method of No.1 was excised, and the results are shown in FIG. 6, in which: the lymph node treated by the method has the advantages of complete tissue structure, normal cell morphology, clear expression and positioning of immunohistochemical protein and clear staining.

The preparation method of the digestive juice comprises the following steps: weighing 1g of EDTA powder, dissolving in PBS, properly adding a few drops of NaOH to adjust the pH value to 8 during preparation, and uniformly stirring until the solution is 100ml to prepare 1% EDTA mother liquor. 2.5g of trypsin and 50mg of proteinase K are respectively weighed and dissolved in 90ml of PBS solution, 1ml of EDTA mother solution is dripped, and PBS is dripped to the total amount of the solution of 100 ml.

The fixative solution is prepared by the following steps: weighing PBS powder and 15ml of distilled water, uniformly mixing to obtain a 7 XPBS solution, then sequentially adding absolute ethyl alcohol, methanol, acetone, formaldehyde, lecithin, deoxycholic acid and methylene blue, fixing the volume to 100ml, and stirring until the mixture is uniformly mixed.

Example 4: control experiment for different treatment methods

Example 1

Fat tissue fixed by conventional formalin (see figure 7) → abandoning the fixing solution, washing by PBS, soaking by 0.25N hydrochloric acid at normal temperature for 1 hour (see figure 8, no obvious change) → abandoning hydrochloric acid, washing by PBS, soaking by 2N NaOH at normal temperature for 1 hour (see figure 9, no obvious change) → abandoning 2N NaOH, washing by PBS, soaking by PBS at 37 ℃ at constant temperature (proteinase K10 mg/ml; proteinase K10 mg/ml + pancreatin 25mg/ml + EDTA 1%; proteinase K5 mg/ml + pancreatin 25mg/ml + EDTA 1%; proteinase K1 mg/ml + pancreatin 25mg/ml + EDTA 1%, forming sample one, two, three, and four in turn from left to right), observing at intervals of one hour (see figure 10, 1h fat tissue pine soft liquefaction; see figure 11, 2h fat tissue soft liquefaction; see figure 12, after 3h, the sample I is dissolved into the sample II, the sample II is further softened, and the sample IV is still in a soft and liquefied state; see fig. 13, after 4h the first sample fat was significantly dissolved, the second, third and fourth samples tissue were partially dissolved) → discarding the digestive juice, and soaking the fat-dissolving fixative for 8 hours (see fig. 14, all tissues were dissolved). Example 2

The fixation and the acid-base pretreatment are carried out in the same way as in example 1 → 2N NaOH is discarded, PBS is soaked in different ways after being washed (normal temperature fixative; 37 ℃, protease K1 mg/ml; 37 ℃, protease K0.5 mg/ml + pancreatin 25mg/ml + EDTA 1%; 37 ℃, protease K0.25 mg/ml + pancreatin 25mg/ml + EDTA 1%; 37 ℃, protease K0.125 mg/ml + pancreatin 25mg/ml + EDTA 1%; samples one, two, three, four, and five are formed in sequence from left to right), observation is carried out every hour (see FIG. 15, 1h shows no obvious change; see FIG. 16, 2h, samples two and three are slightly loose, the rest groups do not obviously change; see FIG. 17, 3h, sample one shows no obvious change, samples two, three, and four are softened compared with two hours, sample five shows no obvious change, see 18, 4h, samples two groups are partially dissolved, sample one, three, and four were softer than at two hours, sample five did not change significantly) → discarding the digestive juice, and soaking in a lipid-dissolving fixative for 8 hours (see fig. 19, left side for sample one, sample four, right side for sample two, and sample two digested for four hours tissue liquefaction).

Example 3

The same example 1 → 2N NaOH is discarded after the fixation and the acid-base pretreatment, 1mg/ml proteinase K is soaked at the constant temperature of 37 ℃ after the PBS is washed, the pH values are different (the pH values are 6, 7, 8 and 9 in sequence, a sample I, a sample II, a sample III and a sample IV are formed in sequence from left to right, the tissue is slightly soft after 3h, the pH value between 6 and 9 has no obvious influence, see figure 20), the digestive juice is discarded, and the fat dissolving fixative is soaked for 8h (see figure 21, the fat dissolving effect is poor).

Example 4

Fixing and acid-base pretreatment are carried out in the same manner as in example 1 → 0.5mg/ml proteinase K + 1% EDTA, soaking at 37 ℃ and constant temperature, adding trypsin with different concentrations (the trypsin concentrations are 12.5mg/ml, 1.25mg/ml and 1.25mg/ml in sequence, and forming a first sample, a second sample, a third sample and a fourth sample in sequence from left to right, observing every hour, wherein after 1 hour, the tissue is hard and does not obviously change as shown in figure 22, and after 2 hours, the fat is loose as shown in figure 23), discarding digestive juice, and soaking a fat dissolving fixing agent for 8 hours (shown in figure 24, the fat is hard and has poor effect).

Example 5

Fixing and acid-base pretreatment are carried out in the same manner as in example 1 → 1mg/ml proteinase K + 1% EDTA at a constant temperature of 37 ℃, trypsin with different concentrations is added (the concentration of the trypsin is 25mg/ml, 2.5mg/ml and 2.5mg/ml in sequence, and a first sample, a second sample, a third sample and a fourth sample are formed in sequence from left to right, and observed for each hour, wherein after 1 hour, the obvious change is not seen in figure 25, and after 2 hours, the fat is loosened in figure 26), the digestive juice is discarded, and the fat-dissolving fixing agent is soaked for 8 hours at room temperature (see figure 27, the fat becomes hard and the effect is poor).

Example 6

The fresh adipose tissues are divided into four samples (see figure 28, sequentially from left to right, a first sample, a second sample, a third sample and a fourth sample) → a first sample and a second sample are soaked in a fixing solution at room temperature for 3 hours, the third sample and the fourth sample are soaked in a Formalin at room temperature for 3 hours (see figure 29, four fats are all hardened) → a first sample and a third sample are treated at room temperature by 0.25N HCl, the second sample and the fourth sample are not treated (see figure 30, hardness: a second sample and a fourth sample > a first sample, hard fat and tough, particles are obvious) → different concentrations of NaOH (see figure 31, the first sample and the third sample are treated by 1mg/ml of protease k +25mg/ml of trypsin-EDTA in a 37 ℃ water bath, and the second sample and the fourth sample are treated by 2N NaOH (see figure 32, the first sample and the third sample have no obvious difference, and are all soft, the second sample and the fourth sample are still treated by 37 ℃ protease k +25mg/ml of trypsin-EDTA in a water bath The first sample and the third sample are not treated (after 9h, see fig. 33, the softening effect of the fat of the first sample and the second sample is better than that of the third sample and the fourth sample, the first sample is not different from that of the second sample and the third sample) → the first sample and the third sample are treated by the fat dissolving fixing solution, the second sample and the fourth sample are not treated (after 10h, see fig. 34, no obvious difference with 9 h) → the second sample and the fourth sample are treated by the fat dissolving fixing solution, and the first sample and the third sample are not treated (after 8h, see fig. 35, the effect of fixing for 4h is better than 3h, and the effect of the fat dissolving fixing solution is better than that of Formalin under the same time).

Example 7

The fresh material taking specimen is divided into four samples (see figure 36, sequentially from left to right, a first sample, a second sample, a third sample and a fourth sample) → a fat dissolving fixing solution soaking at room temperature for 3h (see figure 37, hard tissue) → a first sample 0.25N HCl room temperature treatment, a second sample 2N HCl room temperature treatment, a third sample and a fourth sample non-treatment (see figure 38 after 1h, no obvious change) → a first sample and a fourth sample 2N NaOH treatment, a second sample and a third sample 0.25N NaOH treatment (see figure 39, no obvious difference) → a first sample and a second sample adopt 37 ℃ water bath 1mg/ml proteinase k +25mg/ml trypsin-EDTA treatment, a second sample and a fourth sample non-treatment (after 1h, see figure 40, a first sample fat softening, a second sample fat, a third sample and a fourth fat softening, a first sample fat and a second sample softening, a fibrous tissue appears, a second sample has the best effect, a third sample and a fourth sample softening effect), And (5) treating the sample two with the lipid-dissolving fixing solution, and treating the sample three with the sample four without treatment (after 5h, see fig. 42, almost all the fat in the sample one and the sample two is dissolved, fibrous tissues in the sample three and the sample four appear, and the effect of the sample four is better than that of the sample three) → treating the sample three and the sample four with the lipid-dissolving fixing solution, and treating the sample one and the sample two without treatment (see fig. 43, and the effect of the sample one, the sample two, the sample three and the sample four can reach the expected effect).

Example 8

The fresh material taking samples are divided into ten samples (see fig. 44, the first line is sequentially a first sample, a second sample, a third sample, a fourth sample and a fifth sample from left to right, and the second line is sequentially a sixth sample, a seventh sample, an eighth sample, a ninth sample and a tenth sample from left to right) → the lipid dissolving fixing solution is soaked for 4h (see fig. 45, the hardened fat) → 0.1N HCl/PBS is soaked for 1h at normal temperature (see fig. 46, no obvious change) → 0.1N NaOH/PBS is soaked for 1h at normal temperature (see fig. 47, no obvious difference) → the first sample and the sixth sample K1 mg/ml; sample II, sample heptapancreatin-EDTA 25 mg/ml; sample III, sample octapancreatin-EDTA 12.5mg/ml, enzyme K1 mg/ml; sample four, sample nine pancreatin-EDTA 2.5mg/ml, enzyme K1 mg/ml; sample five and sample ten pancreatin-EDTA 25mg/ml, enzyme K0.5 mg/ml (after 4h, see figure 48, no obvious change) → the fat dissolving stationary liquid soaking for 8h at normal temperature (see figure 49, sample one and sample six compare with sample two and sample seven, the enzyme K effect is better than that of trypsin-EDTA, and sample two and sample seven compare with sample five and sample ten, the combined use effect of the enzymes is better).

Example 9

Soaking the sample lipid-dissolving fixing solution at normal temperature for 3h (see figure 50, the first line is sequentially sample one, sample two, sample three, sample four, sample five, sample six from left to right, and the fat is hardened) → after washing with PBS, soaking with 1N HCl/PBS at room temperature for 1h (see figure 51, no obvious change) → discarding HCl, after washing with PBS, soaking with 0.1N NaOH/PBS at room temperature for 1h (see figure 52, no obvious change) → discarding 0.1N NaOH, after washing with PBS, soaking with constant temperature at 37 ℃ for 3h under the following conditions, (both containing pancreatin 25mg/ml and EDTA 1%), treating with protease K at different concentrations (sample one, sample four 0.5mg/ml, sample two, sample five 0.25mg/ml, sample three, sample six 0.1mg/ml, after 4h, see figure 53, softening the adipose tissue is softened) → sample one, sample two, sample three sample lipid-dissolving fixing solution is soaked for 8h at normal temperature, and formalin is soaked with four, five sample six sample formalin at normal temperature for 8h (see figure 54, the first sample and the fourth sample have good effect, the second sample and the third sample have good effect, and the fifth sample and the sixth sample have poor effect).

In summary, the concentration of each component in the digestive juice is preferably: proteinase K0.1-2 mg/ml (preferably 0.5 mg/ml), trypsin 1-100mg/ml (preferably 25mg/ml), EDTA0.1-2mg/ml (preferably 1 mg/ml).

Claims

1. The application of digestive juice in digesting and liquefying fat of in-vitro tissue specimens for cleaning tumor lymph nodes is characterized in that the digestive juice is composed of the following components in concentration ratio: 0.5mg/ml of proteinase K, 25mg/ml of trypsin and 0.1mg/ml of EDTA; the specimen is a tumor lymph node radical treatment specimen which is fixed by a fixing solution and treated by acid and alkali in sequence, and the fixing solution comprises the following components in concentration ratio: 100ml/L of formaldehyde, 50g/L of lecithin, 250ml/L of absolute ethyl alcohol, 250ml/L of acetone, 47.5g/L of deoxycholic acid, 250ml/L of methanol, 1X of PBS solution and 0.1g/L of methylene blue.

2. The use of the digestive juice according to claim 1 in digesting and liquefying fat from a tumor lymph node cleaning isolated tissue specimen, wherein the digestive juice comprises the following components: the digestion solution also contains a buffer solution which is Tris-HCl with the pH value of 7.5.

3. The use of the digestive juice according to claim 1 in digesting and liquefying fat from a tumor lymph node cleaning isolated tissue specimen, wherein the digestive juice comprises the following components: the digestion solution also contained a buffer solution, which was PBS with pH 7.4.

4. The use of the digestive juice according to claim 3 in digesting and liquefying fat of an isolated tissue specimen for tumor lymph node cleaning, which is characterized in that: the preparation method of the digestive juice comprises the following steps: weighing EDTA powder, dissolving the EDTA powder in PBS, adjusting the pH value of the solution to be proper during preparation, and stirring uniformly to prepare EDTA mother liquor; respectively weighing trypsin and proteinase K, dissolving the trypsin and proteinase K in a buffer solution, then dripping the dissolved enzyme buffer solution into EDTA mother solution in proportion, and supplementing and dripping PBS according to needs.

5. The use of a digestive fluid according to any one of claims 1 to 4 for digesting and liquefying fat in a tumor lymph node clearing isolated tissue specimen, wherein the digestive fluid comprises: CaCl is also added into the digestive juice₂Said CaCl₂The addition amount is 1-10 mM.

6. A method for digesting and liquefying fat of a tumor lymph node cleaning in vitro tissue specimen is characterized in that the specimen is sequentially treated as follows:

(1) treating with the fixing solution at room temperature for 3 hours; (2) treating with 0.1N HCl/PBS solution at room temperature for 1 hour; (3) treating with 0.1N NaOH/PBS solution at room temperature for 1 hr; (4) treating with digestive juice in 37 deg.C water bath for 4 hr;

the fixing solution comprises the following components in concentration ratio: 100ml/L of formaldehyde, 50g/L of lecithin, 250ml/L of absolute ethyl alcohol, 250ml/L of acetone, 47.5g/L of deoxycholic acid, 250ml/L of methanol, 1 x of PBS solution and 0.1g/L of methylene blue;

the digestive juice comprises the following components in concentration ratio: proteinase K0.5 mg/mL, trypsin 25mg/mL, EDTA0.1 mg/mL.

7. The method for digesting and liquefying fat of the tumor lymph node cleaning isolated tissue specimen as claimed in claim 6, wherein: the digestion solution also contains a buffer solution which is Tris-HCl with the pH value of 7.5.

8. The method for digesting and liquefying fat of the tumor lymph node cleaning isolated tissue specimen as claimed in claim 6, wherein: the digestion solution also contained a buffer solution, which was PBS with pH 7.4.

9. The method for digesting and liquefying fat of the tumor lymph node cleaning isolated tissue specimen according to claim 8, wherein the preparation method of the digestive juice comprises the following steps: weighing EDTA powder, dissolving the EDTA powder in PBS, adjusting the pH value of the solution to be proper during preparation, and stirring uniformly to prepare EDTA mother liquor; respectively weighing trypsin and proteinase K, dissolving the trypsin and proteinase K in a buffer solution, then dripping the dissolved enzyme buffer solution into EDTA mother solution in proportion, and supplementing and dripping PBS according to needs.

10. The method for digesting and liquefying fat from the tumor lymph node clearing isolated tissue specimen according to any one of claims 6 to 9, wherein: CaCl is also added into the digestive juice₂Said CaCl₂The addition amount is 1-10 mM.