This method is a guide to use the Elabscience ® RTU antibody for IHC correctly with Benchmark XT (Roche) for immunohistochemical operation and obtain the optimal results.
This method is suitable for the automatic immunohistochemical staining experiment on the Benchmark XT (Roche) instrument with the Elabscience ® RTU antibody for IHC.
3. Experimental Steps
1) Reagents and Consumables
1.1 Transparent dewaxing solution.
1.2 Skim Milk Powder
1.3 Absolute ethanol.
1.4 95% ethanol.
1.5 10× EZ PREP SOLUTION. Cat: 05279771001, Roche.
1.6 Reaction Buffer Concentrate (10×). Cat: 05353955001, Roche.
1.7 LCS (Predilute). Cat: 05264839001, Roche.
1.8 10× SSC SOLUTION Cat: 05353947001, Roche.
1.9 CELL CONDITIONING SOLUTION, CC1 (pH 8.4). Cat: 05279801001, Roche.
1.10 UltraView Universal DAB Detection Kit. Cat: 05269806001, Roche.
1.11 Blue Return Solution: BLUING REAGENT. Cat: 05266769001, Roche.
1.12 Hematoxylin Staining Solution: HEMATOXYLIN II Cat: 05277965001, Roche.
1.13 KIT PACK, EBAR (US/EUROPE). Cat: 05248850001, Roche.
1.14 RIBBON, EBARPRINTER. Cat: 05250889001, Roche.
1.15 Neutral Balsam.
2.1 Automatic Immunohistochemical Staining System: Benchmark XT, Roche.
3) Staining Procedure
3.1. Baking Slice
3.1.1 Select the target tissue slices to be examined, and mark the slices with correct name and markers.
3.1.2 Insert the slices into the dyeing frame and put it into the oven (62°C) to bake the slices for 1 h.
3.2. Print Labels
3.2.1 Click "Barcode Label" to write the label information, print the labels, and paste the labels on the slices.
3.3. Immerse the Slices in 10% Skim Milk Solution
3.3.1 Add 20 g skimmed milk powder into 200 ml distilled water and the mixture is 10% skimmed milk solution.
3.3.2 Immerse the labeled slices in 10% Skim Milk Solution for 15 min to avoid tissue exfoliation.
3.3.3 Wash the slices with tap water to wash most of the 10% skimmed milk solution and wait for the IHC staining.
3.4. Benchmark XT Staining Procedure
3.4.1 Place the slices on the slicing disk, dry the moisture on the slices, and ensure that each slice is placed stably, all of which are in the four snaps of the slicing disk.
3.4.2 Put DAB kit, Hematoxylin Staining Solution, Blue Return Solution and other reagents into the reagent rack (please make sure that the corresponding reagents are sufficient), and check whether the sampling port is full of liquid. If it is found that it is not full of liquid, please squeeze the reagent bottle, exhaust the air, and let the liquid fill the filling port to form a half moon liquid surface.
3.4.3 Set protocol through Ventana nexes software, click Run to complete the pre operation confirmation information. Click START RUN, and the system will conduct self-inspection first, then the program will start to run.
3.4.4 IHC Staining Program
22.214.171.124 Paraffin [Selected].
126.96.36.199 Deparaffinization [Selected].
188.8.131.52 Cell Conditioning [Selected].
184.108.40.206 Conditioner 1 [Selected].
220.127.116.11 Mild CC1 95Deg C and incubate for 30 min [Selected]. (After the completion of antigen repair, the instrument will prompt to pull out the slice table, add the primary antibody, then push the slice table into the instrument, press the prompt key on the instrument manually, and then enter the subsequent staining procedure.)
18.104.22.168 Ab Incubation Temperatures [Selected].
22.214.171.124 Disable Heat for Ab Inc. [Selected].
126.96.36.199 Titration [Selected].
188.8.131.52 Hand Apply (Primary Antibody), and Incubate for 32 min.
184.108.40.206 Counterstain [Selected].
220.127.116.11 Apply One Drop of [HEMATOXYLIN II] (Counterstain), Apply Coverslip, and Incubate for 12 min.
18.104.22.168 Post Counterstain [Selected].
22.214.171.124 Apply One Drop of [BLUING REAGENT] (Post Counterstain), Apply Coverslip, and Incubate for 4 min.
3.4.5 After the staining, take out the slice table, then take out the slices and wash with tap water containing detergent for cleaning. Ensure that the grease on each slice is cleaned, and then wash the slices with running water until they are cleaned, dry the residual water on the slices and enter the subsequent treatment.
3.5. Dehydration and Transparency
3.5.1 Immerse the slices in 75% ethanol for 30 s/time, 2 times.
3.5.2 Immerse the slices in 95% ethanol for 30 s/time, 2 times.
3.5.3 Immerse the slices in absolute ethanol for 30 s/time, 2 times.
3.5.4 Immerse the slices in transparent dewaxing solution for 5 min.
Tips: Drain every time when changing the solution (about 5 s), and then put it into the next solution.
3.6. Seal the Slices
3.6.1 Take out the slices and drain them (about 5 s), blow them dry with cold air by blower.
3.6.2 Add 1~2 drops of neutral balsam to the slice (depending on the number and size of the slice), and cover the slide.
3.6.3 Check the slice to make sure that the slice tissue is completely covered with neutral balsam, and there is no big bubble in the tissue (discharge big bubble by squeezing the cover glass).
3.6.4 Place slices in fume hood to dry.
4). Observe the slice by microscope and judge the result.
1) This method is for Elabscience® RTU Antibody for IHC applicable on the Autostainer Benchmark XT with matched immunohistochemical staining system. Due to the different kinds / targets of Antibody for IHC, the antigen repair methods may differ, please read the manual carefully and strictly follow the manual.
2) This method recommends the varieties / Models / manufacturers of the important related reagents in the process of immunohistochemistry experiment. If users change the corresponding reagents / raw materials, you need to evaluate the equivalence of the alternative varieties and seek the optimal operating procedures.
3) This method is for Elabscience® RTU Antibody for IHC applicable on the Autostainer Benchmark XT with matched immunohistochemical staining system. If the user changes the immunohistochemical staining system, please evaluate the equivalence of the alternative system and find the optimal operation procedure.