Immunostaining of Lymphocytes in Mouse Atherosclerotic Plaque


Target antigen

Host species (label)

Clone

Dilution

Comment

Mouse CD3

Hamster

500A2

1:100

Permeabilization step required

Mouse CD4

Rat

H129.19

1:100
 
Mouse CD8

Rat

53-6.7

1:100
 
Mouse FoxP3

Rat

FJK-16s

1:50

Permeabilization step required

Mouse I-Ab

Mouse (biotinylated)

KH74

1:300
 
Mouse CD19

Rat

1D3

1:100
 



Table 2
Suggested secondary antibodies




























Target antibody

Host species (label)

Cat. no.

Source

Dilution

Rat IgG

Rabbit (biotinylated)

BA-4001

Vector

1:200

Hamster IgG

Goat (biotinylated)

BA-9100

Vector

1:200



Table 3
Suggested streptavidin conjugates




























Target

Conjugates

Cat. no.

Source

Dilution

Biotin

Streptavidin-DyLight 488

SA-5488

Vector

1:400

Biotin

Streptavidin-DyLight 594

SA-5594

Vector

1:400



 


19.

Phosphate-buffered saline (PBS): 1.54 mM KH2PO4, 155.17 mM NaCl and 2.71 mM Na2HPO4, pH 7.4, sterilized by autoclaving.

 

20.

Tepid water.

 





3 Methods



3.1 Sectioning of Aortic Roots and Slide Preparation


In order to optimize the results of the immunostaining, the collection, preservation, and processing of organs should be performed in a standardized way.

1.

After dissection, the hearts should be kept in PBS on ice until the mounting, or be directly mounted in mounting medium and being snap frozen. The embedded hearts should be kept at −80 °C until the cryosectioning is performed.

 

2.

For sectioning of the aortic roots, it is recommended to use the method described by Nicoletti et al. [7]. Briefly, the proximal 800 μm of the aortic root is serially sectioned on a cryostat, with each section 10 μm thick. “Point Zero,” where the aortic root begins, is located where the cusps of the aortic valves start to be visible and is found by sectioning the heart in an upstanding position in a caudal-to-cranial direction. From this point, 10 μm thick sections, starting at the 90–880 μm levels (distance from “Point Zero”) are collected to slides following scheme of Fig. 1. The first 90 μm of sections are used to adjust the angle to get all three cusps visible and aligned.

A318721_1_En_10_Fig1_HTML.gif


Fig. 1
Template for aortic root sectioning and organization of slides

 


3.2 Tissue Fixation




1.

Immerse the slides into fixation reagent for 10 min on ice (see Note 11 ).

 

2.

Remove the slides from fixation reagent and let them dry, at room temperature, for 1 h.

 

3.

After fixation, slides should be stored at −20 °C until staining.

 


3.3 Immunostaining



3.3.1 Immunohistochemistry of Acetone-Fixed Frozen Sections


All steps are conducted in room temperature unless otherwise stated.

1.

Encircle sections on slides with a PAP pen (see Note 12 ).

 

2.

Equilibrate sections with rinsing buffer for 5 min (see Note 13 ).

 

3.

Add Quenching solution for 15–30 min (see Note 3 ).

 

4.

Rinse sections with rinsing buffer, three times for 5 min (see Note 14 ).

 

5.

Incubate sections with avidin solution (from Avidin and Biotin blocking solution, Vector, item 8 in Subheading 2) for 15 min. Rinse briefly after incubation.

 

6.

Incubate sections with biotin solution (from Avidin and Biotin blocking solution, Vector, item 8 in Subheading 2) for 15 min.

 

7.

Rinse with rinsing buffer, three times for 5 min.

 

8.

Block the sections with staining buffer for 30 min. Remove the excess of staining buffer from sections, but do not rinse the slides after the incubation.

 

9.

Add the primary antibody diluted in Staining buffer (see Note 15 ).

 

10.

Incubate the slides overnight at 4 °C, or 1 h at room temperature.

 

11.

Rinse with rinsing buffer, three times for 5 min.

 

Nov 30, 2016 | Posted by in MUSCULOSKELETAL MEDICINE | Comments Off on Immunostaining of Lymphocytes in Mouse Atherosclerotic Plaque

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