Optimized immunofluorescence staining protocol for imaging germinal centers in secondary lymphoid tissues of vaccinated mice

Abstract

Location of immune cells that form the germinal center reaction within secondary lymphoid tissues can be characterized using confocal microscopy. Here, we present an optimized immunofluorescence staining protocol to image germinal center structures in fixed/frozen spleen sections from ChAdOx1 nCoV-19 immunized mice. This protocol can be adapted to identify other cell types within secondary lymphoid tissues. For complete information on the generation and use of this protocol to examine immune responses to the COVID vaccine ChAdOx1 nCoV-19, please refer to Silva-Cayetano et al. (2020).

Keywords: Antibody; Cell Biology; Immunology; Microbiology; Microscopy; Model Organisms.

Graphical abstract

Figure 1

Schematic of the tissue dissection, fixation and embedding

Figure 2

Steps for tissue preparation before cryosectioning (A) Spleen embedded in OCT. (B) 2-propanol cooling bath. (C) Frozen Block in 2-propanol cooling bath. (D) Frozen Block on dry ice before wrapped and stored at −80°C.

Figure 3

Immunofluorescence staining steps (A) Hydrophobic barrier around spleen section. (B) Slides rehydration in a staining jar. (C) Example of humidified chamber containing slides. (D) Dark humidified chamber kept in fridge for 12–16 hours.

Figure 4

Confocal settings using of Zen black software for imaging germinal centers (A) Channels configuration. (B) Scanning parameters configuration. (C) Detectors optimization.

Figure 5

Confocal image of the spleen of ChAdOx1 nCoV-19 immunized 3-month-old mouse A 10 μm splenic section was fixed and immunostained with anti-IgD (green), anti-CD3 (magenta), anti-Ki67 (blue) and anti-CD35 (white) antibodies. The image was tiled to reconstruct an image of the entire spleen. Boxed area focused on the splenic white pulp. The splenic B cell follicle (B) and T cells zone (TCZ) are indicated, germinal centers are indicated with white arrowheads. The scale bar represents 500 μm.

Figure 6

Spleen of ChAdOx1 nCoV-19-immunized mice of the indicated ages Confocal microscopy of spleen from ChAdOx1 nCoV-19 immunized mice where germinal center structures can be easily observed in adult mice (A), but mostly absent in the aged group (B). The scale bars represent 500 μm. IgD⁺ B cell follicle in green, CD3⁺ T cells in magenta, Ki67⁺ cells in blue and CD35⁺ follicular dendritic cells in white. Germinal centers are indicated with white arrowheads.

Figure 7

Germinal Center structure after ChAdOx1 nCoV-19 immunization Representative immunofluorescence of sections from spleens of 3-month-old (A) and 22-month-old (B) ChAdOx1 nCoV-19 immunized mice. Left side shows typical field of view of four channels and right side shows 2D composite image during a germinal center acquisition. The scale bars represent 50 μm. IgD⁺ B cell follicle in green, CD3⁺ T cells in magenta, Ki67⁺ cells in blue and CD35⁺ follicular dendritic cells in white.

Figure 8

Representative images of tissues properly and improperly embedded Confocal images of spleens section showing failed (A) and successful (B) rate of freezing while tissue embedding. Ice crystals formation produce the holes observed in (A). The scale bars represent 500 μm.

Figure 9

Representative sections of correctly and incorrectly cryosectioned tissues Spleen tissue crumbles on anti-roll plate while cryosectioning (A) and a successful flat tissue section obtained (B).