Tissue IHC, IF protocol Tutorial

DAY 1: Preparation and Primary Antibody Application

1. 60 ℃ incubation overnight slide
2. Xylene – 5 min (deparaffinⅠ) , 5 min (deparaffin Ⅱ), 10 min (deparaffin Ⅲ)
3. 100% EtOH – 2 min x 1 time
4. 95% EtOH – 2 min x 2 times
5. Rinse in water – 5 min (wash in running water for 5 minutes in a bucket)
6. Microwave 100 ℃ with citrate buffer in the chamber – (immersion of slide in boiling buffer)
7. Cool down – 30 min (cool down with the slide until the solution is completely cooled)
8. 50 mM NH4Cl in PBS – 30 min
9. 1% BSA, 0.2% gelatin, 0.05% saponin in PBS – 10 min x 3 times
10. Primary antibody diluted in 0.1% BSA, 0.3% Triton X-100 in PBS. Slides are placed in chamber overnight at 4 °C.

• Border around the slide tissue with a PAP Pen to prevent the primary antibody from drying overnight

DAY 2: Secondary Antibody Application and Staining

1. Chamber at room temperature – 60 min
2. Rinse in 0.1% BSA, 0.2% gelatin, 0.05% saponin in PBS – 10 min x 2 times, PBS – 10 min x 1 time
3. Secondary antibody diluted in PBS at room temperature – 60 to 90 min
4. Rinse in PBS – 10 min x 3 times
5. Check the color development with DAB – 1 Slide first, check the time, and apply the same time to the rest of the slides

• (Put the color stop immediately into the water chamber, be careful not to overstain, check 1 slide under a microscope, then proceed with the rest of the staining)

1. Rinse in water – 5 min (wash in running water for 5 minutes in a bucket)
2. Hematoxylin – 30 sec~ 1 min

• (Similar to DAB, after checking the color development of 1 slide, apply the same time and be careful not to over-dye)

1. Rinse in water – 10 min (wash in running water for 5 minutes in a bucket)

DAY 2: Dehydration, Mounting, and Preservation

1. 95% EtOH – 2 min x 2 times
2. 100% EtOH – 2 min x 1 time
3. Xylene – 5 min (dehydrationⅠ) , 5 min (dehydration Ⅱ), 10 min (dehydration Ⅲ)
4. Mount with Mounting Solution (IHC)
5. Keep – slide covered for storage at 4 ℃

Please note that the logical flow of these steps appears to be well-organized, but it is important for users to refer to the manufacturer's protocol for specific antibodies and reagents, as some steps may vary depending on the specific products used.

 

 

 

 

#Iba-1 #brain

 

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DAY1 

 

1.    60 ℃ incubation overnight slide

2.    Xylene – 5 min (deparaffinⅠ) , 5 min (deparaffin Ⅱ), 10 min (deparaffin Ⅲ),

3.    100% EtOH – 2 min x 1 time

4.    95% EtOH – 2 min x 2 time

5.    Rinse in water – 5 min (통에 담아 흐르는 물에 5분간 washing)

6.    Microwave 100 ℃ with citrate buffer in the chamber – (끓는 버퍼에 slide 담금)

7.    Cool down – 30 min (용액이 완전히 식을 때 까지 slide와 함께 cool down)

8.    50mM NH4Cl in PBS – 30 min

9.    1 % BSA, 0.2 % gelatin, 0.05 % saponin in PBS – 10 min x 3 times

10.  Primary antibody diluted in 0.1 % BSA, 0.3 % Triton X-100 in PBS. Slides are placed in chamber overnight at 4 ℃. 

(Border around the slide tissue with a PAP Pen to prevent the primary antibody from drying overnight)

(PAP Pen으로 슬라이드 조직 주위를 테두리 쳐 1차 항체가 overnight 동안 마르지 않게 함)

 

DAY2

11.  Chamber at room temperature – 60 min

12.  Rinse in 0.1 % BSA, 0.2 % gelatin, 0.05% saponin in PBS – 10 min x 2 times,               PBS – 10 min x 1 time

13.  Secondary antibody diluted in PBS at room temperature – 60 ~90 min

14.  Rinse in PBS – 10 min x 3 times

15.  DAB – 1 Slide로 발색 먼저 확인 후 시간을 확인하여 나머지 slide 동일하게 시간 적용 (발색 stop은 water chamber에 바로 넣음, 과염색 되지 않게 주의, 현미경 으로 1 slide check 후 나머지 염색 진행)

16.  Rinse in water – 5 min (통에 담아 흐르는 물에 5분간 washing)

17.  Hematoxylin – 30sec~ 1min (DAB과 동일하게 1 slide 발색 확인 후 동일 시간 적용하여 염색, 과염색 되지않게 주의)

18.  Rinse in water – 10 min (통에 담아 흐르는 물에 5분간 washing)

19.  95% EtOH – 2 min x 2 time

20.  100% EtOH – 2 min x 1 time

21.  Xylene – 5 min (dehydrationⅠ) , 5 min (dehydration Ⅱ), 10 min (dehydration Ⅲ),

22.  Mount with Mounting solusion (IHC)

23.  Keep – slide covered for hoil at 4 ℃

 

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Buffer Solution for Immuno-perosidase-staining (IF, IHC)

 

TEG buffer (pH 9)                          0.01 M PBS (pH 7.4)

Tris : 1.211 g                                   NaH2PO4·H2O : 0.3864 g

EDTA : 0.190 g                               Na2HPO4·2H2O : 1.2815 g

DW : 1 L                                          NaCl : 8.766 g

                                                        DW : 1 L

 

1)    1 % BSA, 0.2 % gelatin, 0.05 % saponin in 0.01 M PBS

BSA : 0.5 g

Gelatin : 0.1 g

Saponin : 0.025 g

è  In 0.01 M PBS 50 ml

 

2)    0.1 % BSA, 0.2 % gelatin, 0.05% saponin in 0.01 M PBS

BSA : 0.05 g

Gelatin : 0.1 g

Saponin : 0.025 g

è  In 0.01 M PBS 50 ml

 

3)    Primary, secondary Ab. Buffer

BSA : 0.005 g

Triton X-100 : 15 ul

è  In 0.01 M PBS 5 ml