Multiplex fluorescence immunohistochemical kit and system for identifying adenocarcinoma and squamous-cell carcinoma by small tissues

Abstract

The invention discloses a multiplex fluorescence immunohistochemical kit and system for identifying adenocarcinoma and squamous cell carcinoma by small tissues. The kit comprises an antibody group and a fluorescent dye group, the antibody group comprises the following components: Synaptysin, ChromograninA, Thyandroid Transcriptional Factor-1, Napsin A, p63, p40, a ChumograninA, a ChumograninA, a ChumograninA, a ChumograninA, a ChumograninA, a ChumograninA, the fluorescent dye group comprises six different fluorescent dyes which are respectively in one-to-one correspondence with the antibodies. According to the detection method disclosed by the invention, multiple fluorescence immunohistochemical staining of the lung cancer small tissue and the puncture type specimen can be realized on the slide of one tissue slice, multiple target molecules can be marked at the same time, the defect that the puncture small tissue cannot be continuously cut for IHC identification and distinguishing can be avoided, and the waste of precious clinical tumor samples is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a multiplex fluorescent immunohistochemistry kit and system for distinguishing adenocarcinoma and squamous cell carcinoma from small tissues. Background Art

[0002] Accurate histopathological and molecular typing play an important role in the selection of treatment options and prognosis of lung cancer patients. Some lung cancer patients are already in the advanced stage when they seek medical treatment and cannot be surgically removed, so diagnosis with small biopsy specimens is particularly important.

[0003] Non-small cell carcinoma is poorly differentiated, and it is difficult to further classify it based on morphological small biopsies alone. Immunohistochemistry is needed to differentiate non-small cell carcinoma into adenocarcinoma and squamous cell carcinoma as much as possible. However, in small biopsy specimens, due to the limitations of sampling and other factors, the existing immunohistochemistry analysis methods are small and precious, and only one target can be marked on a tissue section slide. Several or even dozens of consecutive sections are required to meet the needs of immunohistochemistry multi-target staining for differential diagnosis. Multiple immunofluorescence technology can simultaneously mark multiple target molecules while realizing multiple fluorescent immunohistochemical staining, which can avoid wasting precious clinical tumor samples and save samples. At the same time, the spatial relationship between each target on the same slide can also be analyzed. Summary of the invention

[0004] The purpose of the present invention is to provide a multiplex fluorescent immunohistochemistry kit and system for distinguishing adenocarcinoma and squamous cell carcinoma from small tissues in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect is to provide a multiplex fluorescent immunohistochemistry kit for distinguishing adenocarcinoma and squamous cell carcinoma in small tissues, comprising an antibody group and a fluorescent dye group;

[0007] The antibody group includes neuroendocrine cells and their tumor markers-Synaptophysin, markers for diagnosing neuroendocrine tumors-Chromogranin A, lung adenocarcinoma markers-Thyroid Transcription Factor-1, lung adenocarcinoma markers and lung neuroendocrine tumor negative-NapsinA, lung squamous cell carcinoma markers-p63, lung squamous cell carcinoma markers-p40;

[0008] The fluorescent dye set includes six different fluorescent dyes, each corresponding to the antibody.

[0009] Furthermore, the fluorescent dye group includes Opal 780, Opal 480, Opal 690, Opal 620, Opal 520, and Opal 570 corresponding to Synaptophysin, Chromogranin A, Thyroid Transcription Factor-1, Napsin A, p63, and p40, respectively.

[0010] Furthermore, the preparation method of the primary antibody working solution is as follows: Synaptophysin is diluted at 1:5000, ChromograninA is diluted at 1:10000, Thyroid Transcription Factor-1 is diluted at 1:3000, NapsinA is diluted at 1:4000, p63 is diluted at 1:10000, and p40 is diluted at 1:600.

[0011] Furthermore, the fluorescent dye set also includes a signal amplification solution.

[0012] Furthermore, the multiplex fluorescent immunohistochemistry kit also includes a dewaxing agent, a hydrating agent, sterile deionized water, an antigen repair solution, an antibody diluent, a mounting medium, a buffer, a DAPI working solution, and a blocking solution.

[0013] Furthermore, the antigen repair solution is Tris / EDTA antigen repair solution, pH=9.0; and the buffer solution is 1×TBST buffer solution.

[0014] The second aspect is to provide a system for distinguishing adenocarcinoma and squamous cell carcinoma from small tissues, comprising the above-mentioned multiple fluorescent immunohistochemistry kit and a pathological image acquisition and analysis system adapted thereto; pathological images are obtained by performing multiple fluorescent immunohistochemistry operations on biological samples; the pathological image acquisition and analysis system acquires images of the pathological images and analyzes the images based on the following markers: Synaptophysin, Chromogranin A, Thyroid Transcription Factor-1, Napsin A, p63, and p40.

[0015] Furthermore, when performing immunohistochemistry, after the specimen is dewaxed and hydrated, the antigen retrieval and staining round operating conditions of each antibody in the primary monoclonal antibody group are as follows:

[0016] In the first round, the repair time was 15 min, the blocking time was 10 min, the primary antibody incubation with Chromogranin A was 45 min, the rinsing time was 10 min, the secondary antibody incubation with HRP was 10 min, the rinsing time was 10 min, and the 480 dye incubation time was 10 min;

[0017] In the second round, the repair time was 15 min, the blocking time was 10 min, the incubation time was 45 min with the primary antibody NapsinA, the rinsing time was 10 min, the secondary antibody incubation was HRP for 10 min, the rinsing time was 10 min, and the incubation time was 620 for 10 min;

[0018] In the third round, the repair time was 15 min, the blocking time was 10 min, the P63 primary antibody was incubated for 45 min, the rinsing time was 10 min, the secondary antibody was incubated with HRP for 10 min, the rinsing time was 10 min, and the 520 dye was incubated for 10 min;

[0019] In the fourth round, the repair time was 15 min, blocking was 10 min, incubation with Thyroid Transcription Factor-1 primary antibody, incubation with 690 for 45 min, rinsing for 10 min, incubation with HRP secondary antibody for 10 min, rinsing for 10 min, and incubation with 690 dye for 10 min;

[0020] In the fifth round, the repair time was 15 min, the blocking time was 10 min, the P40 primary antibody was incubated for 45 min, the rinsing time was 10 min, the secondary antibody was incubated with HRP for 10 min, the rinsing time was 10 min, and the 570 dye was incubated for 10 min;

[0021] In the sixth round, the repair time was 15 min, the blocking time was 10 min, the primary antibody incubation with Synaptophysinp was 45 min, the rinsing time was 10 min, the secondary antibody incubation with HRP was 10 min, the rinsing time was 10 min, the DIG incubation time was 10 min, the rinsing time was 10 min, and the 780 dye incubation time was 60 min; the rinsing time was 10 min;

[0022] The seventh round: DAPI, 10 min, rinse for 10 min, and seal the slides.

[0023] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0024] The present invention is based on multiple fluorescent immunohistochemistry technology, processes and analyzes the staining results of multiple markers of lung cancer, uses an imaging microscope to collect spectral images, and uses case picture analysis software to identify the images, so that small lung cancer tissues and puncture type specimens can achieve multiple fluorescent immunohistochemical staining on a tissue section slide, and can mark multiple target molecules at the same time, which can avoid the disadvantage that punctured small tissues cannot be continuously cut for IHC identification and differentiation, and avoid wasting precious clinical tumor samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1The expression of P40-480 dye in scheme A is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0026] Figure 2 The expression of Napsin A-570 dye in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma in scheme A is shown.

[0027] Figure 3 The expression of TTF-1-690 dye in scheme A is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0028] Figure 4 The expression of Syn-520 dye in scheme A is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0029] Figure 5 The expression of CgA-620 dye in scheme A in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma is shown.

[0030] Figure 6 The expression of P63-780 dye in scheme A is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0031] Figure 7 The expression of CgA-480 dye in scheme B is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0032] Figure 8 The expression of NapsinA-620 dye in scheme B is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0033] Fig. 9 The expression of P63-520 dye in scheme B is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0034] Fig.10 The expression of TTF-1-690 dye in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma in protocol B is shown.

[0035] Fig.11 The expression of P40-570 dye in scheme B is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0036] Fig.12 The expression of Syn-780 dye in scheme B is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0037] Fig.13 The expression of Syn-690 dye in scheme C is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0038] Fig.14 The expression of TTF1-570 dye in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma in scheme C is shown.

[0039] Fig.15The expression of CgA-620 dye in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma in scheme C is shown.

[0040] Fig.16 The expression of P63-480 dye in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma in scheme C is shown.

[0041] Fig.17 The expression of Napsin A-520 dye in scheme C is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma.

[0042] Fig.18 The expression of P40-780 dye in scheme C is shown in pancreas, lung squamous cell carcinoma, and lung adenocarcinoma. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0044] The reagents and raw materials used in the following examples are all commercially available or can be prepared according to literature methods. The experimental methods in the following examples without specifying specific conditions are generally carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by volume.

[0045] 1. Detection markers

[0046] Neuroendocrine cells and their tumor markers-Synaptophysin (Syn)

[0047] Chromogranin A (CgA) is a marker for the diagnosis of neuroendocrine tumors

[0048] Lung Adenocarcinoma Marker - Thyroid Transcription Factor-1(TTF1)

[0049] Lung adenocarcinoma marker, negative for pulmonary neuroendocrine tumors - Napsin A

[0050] Lung squamous cell carcinoma marker-p63

[0051] Lung squamous cell carcinoma marker-p40

[0052] II. Materials and Reagents of Multiplex Fluorescence Immunohistochemistry Kit

[0053] The reagents used in the multiplex fluorescent immunohistochemistry technique are as follows: Opal 480, Opal 520, Opal 570, Opal 620, Opal 690, Opal 780, DAPI, immunochromogenic reagent strips (RF029100), dewaxing agent, hydrating agent, sterile deionized water, antigen retrieval solution, TSA fluorescent kit, primary antibody, antibody diluent, mounting medium, washing buffer, and DAPI working solution.

[0054] The preparation process of the above reagents is as follows:

[0055] a) Deionized water sterilization: prepare 1800 mL of deionized water and dispense it into 6 bottles, with the liquid volume less than 2 / 3, 300 mL / bottle, mark the sterilization date, and sterilize at 0.15 MPa and 121°C for 30 minutes;

[0056] b) Xylene: analytical grade, used as stock solution;

[0057] c) Gradient concentrations of 100%, 95%, and 75% ethanol: 100% ethanol was directly used from 100 mL of the original anhydrous ethanol; 95% ethanol was prepared from 5 mL of sterile deionized water and 95 mL of anhydrous ethanol; 75% ethanol was prepared from 25 mL of sterile deionized water and 75 mL of anhydrous ethanol;

[0058] d) Antigen retrieval solution: 20 mL of Tris / EDTA stock solution was dissolved in 1000 mL of ultrapure water, the pH value of the solution was monitored with a pH test paper, and the pH was adjusted to 9.0 (usually with hydrochloric acid) to obtain an antigen retrieval solution;

[0059] e) 1× TBST buffer: Mix 50 mL of 20-fold stock solution with 950 mL of sterile deionized water to prepare 1000 mL of 1× TBST buffer and bottle it for later use;

[0060] f) Blocking solution: The blocking solution provided with the TSA fluorescence kit can be used directly as the working solution;

[0061] g) Primary antibodies: Synaptophysin (Syn), a marker for neuroendocrine cells and their tumors; Chromogranin A (CgA), a marker for the diagnosis of neuroendocrine tumors; Thyroid Transcription Factor-1 (TTF1), a marker for lung adenocarcinoma; NapsinA, a marker for lung adenocarcinoma and negative for lung neuroendocrine tumors; p63, a marker for lung squamous cell carcinoma; p40, a marker for lung squamous cell carcinoma. All antibodies were diluted to the corresponding multiples with antibody diluent according to the antibody instructions to obtain the primary antibody working solution. The specific dilution multiples are as follows: syn (1:5000), CgA (1:10000), TTF1 (1:3000), NapsinA (1:4000), P63 (1:10000), P40 (1:600).

[0062] h) Secondary antibody: TSA fluorescence kit equipped with HRP secondary antibody can be used directly as working solution;

[0063] I) TSA dye: The fluorescent dye working solution is prepared by diluting the mother solution of various fluorescent dyes 100 times in the signal amplification solution and preparing it before use;

[0064] J) DAPI working solution: TSA fluorescence kit, DAPI working solution is diluted 100 times in deionized water and prepared before use.

[0065] 3. Experimental steps

[0066] 1) Dewaxing and hydration:

[0067] The slides with specimens were placed in three portions of xylene in sequence, each for 5 minutes to complete dewaxing; then placed in two portions of 100% ethanol in sequence, each for 5 minutes; then placed in 95% ethanol for 3 minutes, and finally placed in 75% ethanol for 2 minutes, and washed with sterile deionized water 3 times, each for 1 minute, to complete hydration;

[0068] 2) First antigen retrieval:

[0069] Pour antigen repair solution into the repair cup, boil it on high heat in a microwave oven (about 3-4 minutes, subject to actual measurement), put the slide to be repaired in the cup and cook on low heat for 15 minutes, then take it out and cool it naturally to room temperature;

[0070] 3) Closure:

[0071] The repaired slides were rinsed once with 1×TBST buffer, excess water was shaken off, the tissue was circled with a histochemical pen, nonspecific protein blocking agent was added, and the slides were incubated at room temperature (room temperature refers to about 25°C, and moisturizing refers to maintaining appropriate humidity, which is understood in the conventional way, the same below) with shaking for 10 minutes;

[0072] 4) Primary antibody incubation:

[0073] Discard the blocking solution, add the primary antibody working solution diluted with diluent, incubate at room temperature with moisturizing and shaking for 30-60 minutes (can be adjusted according to the specificity of the antibody), and rinse 3 times with 1×TBST buffer, 2 minutes each time;

[0074] 5) Secondary antibody incubation: discard the washing solution, add the secondary antibody working solution, incubate at room temperature with moisturizing and shaking for 10 minutes, and rinse 3 times with 1× TBST buffer, 2 minutes each time;

[0075] 6) TSA incubation staining:

[0076] Discard the washing solution, add the fluorescent dye working solution corresponding to the primary antibody in TSA dye, incubate at room temperature with moisturizing and shaking for 10 minutes, rinse with 1× TBST buffer for 2 minutes, and wash 3 times;

[0077] 7) Repeat steps (2-6) for the next round of antibody staining until all staining of one antibody group in the primary antibody is completed;

[0078] 8) Nuclear staining (DAPI)

[0079] Add DAPI working solution, incubate at room temperature for 10 minutes, and rinse 3 times with 1× TBST buffer, 2 minutes each time;

[0080] 9) Sealing: discard the washing solution, rinse with 1×TBST buffer for 2 minutes, and then wash with sterile deionized water for 3 minutes. After the tissue is slightly dry, add water-soluble anti-fluorescence quenching sealing agent to seal the slide, and fix the coverslip with colorless nail polish.

[0081] 4. Condition Optimization

[0082] Three schemes were set up to test whether the expression of each antibody in different combinations was consistent with the IHC results according to the antibody-dye combination, mainly observing the fluorescence intensity, protein localization and specific staining of each target.

[0083] 4.1 Scheme A:

[0084] The corresponding fluorescence channels of each antibody are as follows:

[0085] P40 antibody corresponds to the 480nm fluorescence channel

[0086] Napsin A antibody corresponds to the 570nm fluorescence channel

[0087] TTF1 antibody corresponds to the 690nm fluorescence channel

[0088] Syn antibody corresponds to the 520nm fluorescence channel

[0089] CgA antibody corresponds to the 620nm fluorescence channel

[0090] P63 antibody corresponds to 780nm fluorescence channel

[0091] The staining rounds for protocol A are determined as follows:

[0092] First round: P40

[0093] Round 2: Napsin A

[0094] Round 3: TTF1

[0095] Round 4: Syn

[0096] Round 5: CgA

[0097] Round 6: P63

[0098] Round 7: DAPI

[0099] 4.2 Scheme B:

[0100] The corresponding fluorescence channels of each antibody are as follows:

[0101] CgA antibody corresponds to the 480nm fluorescence channel

[0102] Napsin A antibody corresponds to the 620nm fluorescence channel

[0103] P63 antibody corresponds to the 520nm fluorescence channel

[0104] TTF1 antibody corresponds to the 690nm fluorescence channel

[0105] P40 antibody corresponds to the 570nm fluorescence channel

[0106] Syn antibody corresponds to 780nm fluorescence channel

[0107] The staining rounds for Scheme B are determined as follows:

[0108] Round 1: CgA

[0109] Round 2: Napsin A

[0110] Round 3: P63

[0111] Round 4: TTF1

[0112] Round 5: P40

[0113] Round 6: Syn

[0114] Round 7: DAPI

[0115] 4.3 Scheme C:

[0116] The corresponding fluorescence channels of each antibody are as follows:

[0117] Syn antibody corresponds to 690nm fluorescence channel

[0118] TTF antibody on 570nm fluorescence channel

[0119] CgA antibody corresponds to the 620nm fluorescence channel

[0120] P63 antibody corresponds to the 480nm fluorescence channel

[0121] Napsin A antibody corresponds to the 520nm fluorescence channel

[0122] P40 antibody corresponds to 780nm fluorescence channel

[0123] The staining rounds for Scheme C are determined as follows:

[0124] Round 1: Syn

[0125] Round 2: TTF1

[0126] Round 3: CgA

[0127] Round 4: P63

[0128] Round 5: Napsin A

[0129] Round 6: P40

[0130] Round 7: DAPI

[0131] Experimental results reference Figure 1-18 The results showed that in scheme B, the antibody-dye combination was the best, the target was clearly located, there was no nonspecific coloring, and the expression level was consistent with the immunohistochemistry results.

[0132] 5. Finalize the staining rounds of Scheme B

[0133] The specific experimental conditions are shown in Table 1:

[0134] Table 1

[0135]

[0136]

[0137] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A multiplex fluorescent immunohistochemistry kit for distinguishing adenocarcinoma and squamous cell carcinoma in small tissues, characterized in that: Includes antibody group and fluorescent dye group; The antibody group includes neuroendocrine cells and their tumor markers-Synaptophysin, markers for diagnosing neuroendocrine tumors-Chromogranin A, lung adenocarcinoma markers-Thyroid Transcription Factor-1, lung adenocarcinoma markers and lung neuroendocrine tumor negative-NapsinA, lung squamous cell carcinoma markers-p63, lung squamous cell carcinoma markers-p40; The fluorescent dye set includes six different fluorescent dyes, each corresponding to the antibody.

2. The multiplex fluorescent immunohistochemistry kit according to claim 1, characterized in that: The fluorescent dye group includes Opal 780, Opal 480, Opal 690, Opal 620, Opal 520 and Opal 570 corresponding to Synaptophysin, Chromogranin A, Thyroid Transcription Factor-1, Napsin A, p63 and p40 respectively.

3. The multiplex fluorescent immunohistochemistry kit according to claim 1, characterized in that: The preparation method of the primary antibody working solution is as follows: Synaptophysin is diluted at 1:5000, Chromogranin A is diluted at 1:10000, ThyroidTranscription Factor-1 is diluted at 1:3000, NapsinA is diluted at 1:4000, p63 is diluted at 1:10000, and p40 is diluted at 1:

600.

4. The multiplex fluorescent immunohistochemistry kit according to claim 1, characterized in that: The fluorescent dye set also includes a signal amplification solution.

5. The multiplex fluorescent immunohistochemistry kit according to claim 3, characterized in that: It also includes dewaxing agent, hydrating agent, sterile deionized water, antigen repair solution, antibody diluent, mounting medium, buffer, DAPI working solution, and blocking solution.

6. The multiplex fluorescent immunohistochemistry kit according to claim 5, characterized in that: The antigen repair solution is Tris / EDTA antigen repair solution, pH=9.0; the buffer is 1×TBST buffer.

7. A system for distinguishing adenocarcinoma and squamous cell carcinoma in small tissues, characterized in that: The method comprises the multiplex fluorescent immunohistochemistry kit as described in any one of claims 1 to 6 and a pathological image acquisition and analysis system adapted thereto; a pathological image is obtained by performing multiplex fluorescent immunohistochemistry operations on a biological sample; the pathological image acquisition and analysis system acquires an image of the pathological image and analyzes the image based on the following markers: Synaptophysin, Chromogranin A, Thyroid Transcription Factor-1, Napsin A, p63, and p40.

8. The system for distinguishing adenocarcinoma and squamous cell carcinoma from small tissues according to claim 7, characterized in that: When performing immunohistochemistry, after the specimen is dewaxed and hydrated, the antigen retrieval and staining rounds of each antibody in the primary monoclonal antibody group are as follows: In the first round, the repair time was 15 min, the blocking time was 10 min, the primary antibody incubation with Chromogranin A was 45 min, the rinsing time was 10 min, the secondary antibody incubation with HRP was 10 min, the rinsing time was 10 min, and the 480 dye incubation time was 10 min; In the second round, the repair time was 15 min, the blocking time was 10 min, the primary antibody NapsinA was incubated for 45 min, the rinsing time was 10 min, the secondary antibody HRP was incubated for 10 min, the rinsing time was 10 min, and the 620 incubation time was 10 min; In the third round, the repair time was 15 min, the blocking time was 10 min, the P63 primary antibody was incubated for 45 min, the rinsing time was 10 min, the secondary antibody was incubated with HRP for 10 min, the rinsing time was 10 min, and the 520 dye was incubated for 10 min; In the fourth round, the repair time was 15 min, the blocking time was 10 min, the primary antibody incubation of ThyroidTranscriptionFactor-1 was 45 min, the 690 incubation time was 10 min, the secondary antibody incubation of HRP was 10 min, the rinsing time was 10 min, and the 690 dye incubation time was 10 min; In the fifth round, the repair time was 15 min, the blocking time was 10 min, the P40 primary antibody was incubated for 45 min, the rinsing time was 10 min, the secondary antibody was incubated with HRP for 10 min, the rinsing time was 10 min, and the 570 dye was incubated for 10 min; In the sixth round, the repair time was 15 min, the blocking time was 10 min, the primary antibody incubation with Synaptophysinp was 45 min, the rinsing time was 10 min, the secondary antibody incubation with HRP was 10 min, the rinsing time was 10 min, the DIG incubation time was 10 min, the rinsing time was 10 min, and the 780 dye incubation time was 60 min; the rinsing time was 10 min; The seventh round: DAPI, 10 min, rinse for 10 min, and seal the slides.

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