Lab Project

LAB PROJECT: IMMUNOHISTOCHEMISTRY (original from Bryan)

Objective: To use immunohistochemistry to identify the location of specific molecules and/or structures in organ sections.

1) Every group will use the naturally fluorescent probe, DAPI to stain cell nuclei. In addition, your group should select 2 antibodies or probes to stain the frozen sections of your organ. Be sure to consider the possible secondary antibodies and their fluorochromes when making your selection; one should be marked with a red fluorochrome (rhodamine) and the other with a green fluorochrome (FITC). Predict what the markers will stain in your particular sections. (ex: anti-laminin will stain basal lamina, etc.) Review your staining plan with a TA before proceeding.

2) Get 2 slides of your frozen sections. Label the slides with your group number. Sections should be on the frosted side of the slide with your group number. Flip the slide over so that the reverse side is facing you. On this side of the slide, use a marker to circle the sections. Doing this will enable you to see where your sections are located in order to stain them

3) Fix slides in the fixative, 1% Formalin for 5 minutes. You must prevent your slides from drying from this point on.

4) Rinse slides in Phosphate-buffered saline (PBS) for 5 minutes on a stir plate in an inverted coplin jar with a magnetic stirrer.

5) Dry the edges of the slides outside the hydrophobic barrier with a Kimwipe, taking care not to touch your sections. Use the Pap Pen to make a hydrophobic barrier on either side of your sections (see Figure above). Place these slides in the humidified staining box.

6) On these slides, use a pipet to place enough blocking solution on the sections to cover them. Let sit for 5 minutes.

7) Drain blocking solution from the slides by tipping them on to some Kimwipes and place 100 ul of your primary staining solution on the sections of each slide. Let sit for 45 minutes. While waiting, stain your other immunohistochemistry slides with toluidine blue (see step 12 below) and/or start making your paraffin sections if you haven’t already.

8) Dip incubated slides in fresh PBS to remove the staining solution, then dump this PBS as well. Rinse the slides for 25 minutes in a new PBS solution, as before.

9) Wipe edges of slides carefully and return them to the staining box. Place 100 ul of your secondary staining solution on the sections of each slide. Let sit for 45 minutes in a humidified box.

10) Again, dip slides in fresh PBST to remove the staining solution, then dump this PBS as well. Rinse the slides for 25 minutes in a new PBS solution, as before.

11) Wipe edges of slides and place one drop of mounting solution on sections. The mounting solution contains DAPI, the cell nuclei marker. Place coverslip on slide. Fix in place with nail polish.

12) Toluidine Blue staining: Fix and rinse the other slides following steps 3-5 above. Place a drop of toluidine blue solution on the sections. After ~15 seconds, rinse the slide and look at it under low power using your microscope. If it is not well stained, stain again. If it is over stained (too dark) use another slide and start staining again for a shorter time. Consult with Bryan.

13) Image your fluorescent and toluidine blue-stained slides.

Antibody Staining

Primary Antibodies

Antibody Name Antigen recognized Produced in Dilution
M7523-.2ML ANTI-SKELETAL MYOSIN Rabbit 1:20
C3595-.2ML ANTI-CONNEXIN 32 Rabbit 1:400
A2668-.2ML ANTI-ACTIN Rabbit 1:40
L9393-.2ML ANTI-LAMININ Rabbit 1:25
DHB 2E8 (IgG2a) Laminin Mouse 1:2
DHB Ralph 3.1 (IgG2a) Integrin, alpha-3 Mouse 1:2
DHB M3F7 (IgG1) Collagen Type IV Mouse 1:2
DHB II-II6B3 (IgG1) Collagen type II Mouse 1:2
DHB JLA20 (IgM) Actin Mouse 1:2
DHB SV2 Synaptic Vesicles Mouse 1:2
DHB TI-4 Anti-Troponin I Mouse 1:2
DHB B4-78 Anti-Alkaline Phosphatase Mouse 1:2
DHB T14 Anti-Myosin Light Chain Mouse 1:2

Secondary Antibodies

Antibody Name Produced in against Dilution
F0382-1ML FITC CONJUGATE GOAT ANTI-RABBIT IGG 1:80
T5393-.5ML T5393 Goat Anti-Mouse IgG 1:64
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