Tomato Lectin Histochemistry for Microglial Visualization

Abstract

The use of different lectins for the study of microglial cells in the central nervous system (CNS) is a valuable tool that has been extensively used in the last years for the selective staining of this glial cell population, not only in normal physiological conditions, but also in a wide range of pathological situations where the normal homeostasis of the parenchyma is disturbed. In this chapter we accurately describe the methodology for the selective labelling of microglial cells by using the tomato lectin (TL), a protein lectin obtained from Lycopersicum esculentum with specific affinity for poly-N-acetyl lactosamine sugar residues which are found on the plasma membrane and in the cytoplasm of microglia. Here we describe how to perform this technique on vibratome, frozen, and paraffin sections for optical microscopy, as well as for transmission electron microscopy (TEM) studies. Using this methodology it is possible to visualize amoeboid microglia in the developing brain, ramified microglia in the adult, and activated/reactive microglia in the experimentally damaged brain. In addition, as TL also recognized sugar residues in endothelial cells, this technique is very useful for the study of the relationship established between microglia and the CNS vasculature.

Reacquisition of cocaine conditioned place preference and its inhibition by previous social interaction preferentially affect D1-medium spiny neurons in the accumbens corridor

“…After a TBS-T wash, slices were incubated for 30 min in TBS containing 50 mM glycine, followed by another wash in TBS-T (3 × 5 min) and a 1 h incubation in TBS-T containing 2% BSA and 10% normal donkey serum (Millipore, S30) or 7% normal donkey and 3% normal goat serum (Vector Labs, S-1000) depending on the primary antibodies used. For the investigation of the involvement of neuronal vs. glial cells in the observed behavioral changes, we performed double immunohistochemistry for EGR1 and a mouse polyclonal antibody against the neuron-specific nuclear protein NeuN (Mullen et al, 1992, anti-NeuN, 1:200, Millipore, MAB377) or one of the following glial markers: Mouse polyclonal anti glial fibrillary acidic protein (anti-GFAP; 1:200, Santa Cruz Biotechnology, sc-33673) to identify astrocytes (Wang et al, 2013), mouse polyclonal myelin basic protein (anti-MBP; 1:200, Santa Cruz Biotechnology, sc-71546) for oligodendrocytes (Najm et al, 2013), or simple tomato lectin staining (6 μg/μl, 24 h incubation, Vector laboratories, DL-1177) for microglia (Villacampa et al, 2013). Sections were washed in 50 mM TBS-T for 1 h and incubated for 2 h in 50 mM TBS-T containing 2% BSA, the anti-rabbit Alexa Fluor 488-conjugated secondary antibody (1:400, Invitrogen, A21441) and the anti-donkey Alexa Fluor 555-conjugated secondary antibody (1:400, Invitrogen, A21432) for DYN, PV, CR and NPY staining or anti-mouse Alexa Fluor 555-conjugated secondary antibody (1:400, Invitrogen, A31570) for DRD2, NeuN, GFAP and MBP staining.…”

Glia–neuron interactions in the mammalian retina

“…Histochemical detection also offers advantages in some cases. For instance, one histochemical method uses a tomato lectin with affinity for sugar moieties present on microglial and endothelial cells, allowing the relationship of microglia with blood vessels to be analysed (Villacampa et al, 2013). Microglia in different activation states can be characterized using combinations of techniques aimed at analyzing the secretory, proteomic and transcriptional signatures (Glanzer et al, 2007): microarrays, real-time RT-PCR (reverse transcription polymerase chain reaction), SELDI-TOF (surface-enhanced laser desorption ionization-time of flight), twodimensional gel electrophoresis (2D-PAGE), mass spectrometry, quantitative western blotting…etc.…”

The hippocampal neurovascular niche during normal development and after irradiation to the juvenile mouse brain

“…Th e SGZ was defi ned as a six-cell layer thick structure corresponding to three cell layers into the hilus and granule cell layer (GCL) from the border, respectively (one cell diameter defi ned as 6 μ m). Tomato lectin is a marker used for both the identifi cation of EC (Persson et al 2010) and microglia (Villacampa et al 2013). In this study we used tomato lectin to visualize blood vessels and excluded microglia based on morphology.…”