Usage Method of Elabscience® RTU Antibody for IHC (Dako Autostainer Link 48)
2019-12-25Author:adminpraise：1
1. Target
This method is a guide to use the Elabscience ® RTU antibody for IHC correctly with Autostainer link 48 (Dako) for immunohistochemical operation and obtain the optimal results.
2. Limits
This method is suitable for the automatic immunohistochemical staining experiment on the Autostainer link 48 (Dako) instrument with the Elabscience ® RTU antibody for IHC.
3. Experimental Steps
1) Reagents and Consumables
1.1 Transparent dewaxing solution.
1.2 Absolute ethanol.
1.3 95% ethanol.
1.4 PBS Buffer.
1.5 Tween-20.
1.6 Immunohistochemical oil pen.
1.7 EnVision™ TMFLEX+, High pH (Link) Cat: K800221-2CN, Dako.
1.8 DAB Away Cat: S196730-2, Dako.
1.9 User Fillable Reagent Bottle, 12 mL Capacity, (Link) Cat: SK20110- 2, Dako.
1.10 User Fillable Reagent Bottle, 5 mL Capacity, (Link) Cat: SK20005-2, Dako.
1.11 Large Flap Slide Label Kit (875×950) Cat: S341730, Dako.
1.12 Hematoxylin Staining Solution.
1.13 Neutral Balsam.
2) Instruments
2.1 Oven.
2.2 PT Link: PT200, Dako.
2.3 Automatic Immunohistochemical Staining System: Autostainer Link 48, Dako.
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 Deparaffinizing and Rehydration
3.2.1 Immerse the slices in transparent dewaxing solution for 15 min/time, 2 times.
3.2.2 Immerse the slices in absolute ethanol for 5 min/time, 2 times.
3.2.3 Immerse the slices in 95% ethanol for 5 min/time, 2 times.
3.2.4 Immerse the slices in 75% ethanol for 5 min/time, 2 times.
Tips: Drain every time when changing the solution (about 5 s), and then put it into the next solution.
3.2.5 Take out the dyeing frame with slices, Drain away the solution. Washing with water for 10 s/time, more than 3times.
3.3 Antigen Retrieval
3.3.1 According to the manual, choose EnVision™ FLEX Target Retrieval Solution High pH (50×), and repair the antigen with PT Link as below.
3.3.2 Take appropriate amount of antigen repair solution into the repair tank, preheat to 75°C in advance.
3.3.3 Immerse the dyeing frame with slices into the repair solution of the repair tank immediately, cover the tank, heat to 97°C, and incubate for 30 minutes.
3.3.4 Wait until the Retrieval Solution is cold about 75°C, uncover the tank, take out of the dyeing frame with slices, Immerse them in Wash Buffer to chill-down.
3.3.5 After the section is cooled, remove the liquid from the slices. Place the dyeing frame on the absorbent paper, draw a circle 2-3 mm away from the tissue with an immunohistochemical oil pen on each slice, then place the slices on the slice frame of Aautostainer link 48.
3.4 For the Autostainer Link 48 staining procedure, choose the FLEX program. The main parameters are as follows
3.4.1 FLEX Peroxidase Block (SM801), Volume (µl): 100, Incubation: 5 min.
3.4.2 Elabscience® RTU Antibody for IHC, Volume (µl): 100, Incubation: 30 min.
3.4.3 FLEX /HRP (SM802), Volume (µl): 100, Incubation: 30 min.
3.4.4 FLEX DAB+ Sub-Chromo (SM803), Volume (µl): 200, Incubation: 10 min.
3.4.5 Refer to flex procedure for other dyeing procedure parameters.
3.4.6 Elabscience® RTU Antibody for IHC, P40 Monoclonal Antibody (Cat: PA6665)
Reagents and Incubation
|
Code |
Reagent name |
Volume(µL) |
Incubation |
|
|
Buffer |
|
0 |
|
SM801 |
FLEX Peroxidase Block |
100 |
5 |
|
|
Buffer |
|
0 |
|
|
RUO-p40 |
100 |
30 |
|
|
Buffer |
|
0 |
|
SM802 |
FLEX/HRP |
100 |
30 |
|
|
Buffer |
|
0 |
|
|
Buffer |
|
5 |
|
SM803 |
FLEX DAB+ Sub-Chromo |
200 |
10 |
|
|
Buffer |
|
0 |
|
|
DI Water |
|
0 |
Staining Procedure
|
Slide log |
|
|
11/19/2019 11:53:51 AM |
Staining started |
|
11/19/2019 11:53:51 AM |
Run ID:11 |
|
11/19/2019 11:54:28 AM |
Rinsed with buffer |
|
11/19/2019 11:58:07 AM |
Blowandapply 100 µL EnVision™ FLEX Peroxidase-Blocking Reagent. |
|
11/19/2019 12:03:10 PM |
Rinsed with buffer |
|
11/19/2019 12:16:04 PM |
Blow and apply 100 µL RUO-p40 |
|
11/19/2019 12:46:04 PM |
Rinsed with buffer |
|
11/19/2019 12:54:37 PM |
Blow and apply 100 µL EnVision™ FLEX/HRP |
|
11/19/2019 1:24:39 PM |
Rinsed with buffer |
|
11/19/2019 1:29:49 PM |
Rinsed with buffer |
|
11/19/2019 1:37:50 PM |
Blow and apply 200uL Substrate Working Solution (mix) |
|
11/19/2019 1:47:56 PM |
Rinsed with buffer |
|
11/19/2019 1:58:27 PM |
Rinsed with water |
|
11/19/2019 1:58:27 PM |
Staining completed |
3.5 Hematoxylin stain the nucleus
3.5.1 After the Autostainer Link 48 staining , take the slices out of the slice frame and put them on the dyeing frame, wash with water for 10 s/time, 2~3 times, Remove the liquid from the dyeing frame, put them into the hematoxylin dyeing solution, and incubate for 7 min.
3.5.2 Take out the slices and drain them (about 5 s), wash them with water to remove the superfluous dye, put the slices into PBST for 60 s, then wash with water for 10 s/time, 3 times, then remove the water from the dyeing frame.
3.6 Dehydration and transparency
3.6.1 Immerse the slices in 75% ethanol for 30 s/time, 2 times.
3.6.2 Immerse the slices in 95% ethanol for 30 s/time, 2 times.
3.6.3 Immerse the slices in absolute ethanol for 30 s/time, 2 times.
3.6.4 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.
3.7 Seal the slices
3.7.1 Take out the slices and drain them (about 5 s), blow them dry with cold air by blower.
3.7.2 Add 1~2 drops of neutral balsam to the slice (depending on the number and size of the slice), and cover the slide.
3.7.3 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).
3.7.4 Place slices in fume hood to dry.
4). Observe the slice by microscope and judge the result.
4. Notices
1) This method is for Elabscience® RTU Antibody for IHC applicable on the Autostainer Link 48 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 Link 48 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.