Antibody incubation at 37°C improves fluorescent immunolabeling in free-floating thick tissue sections

Abstract

Fluorescent immunolabeling and imaging in free-floating thick (50-60 μm) tissue sections is relatively simple in practice and enables design-based non-biased stereology, or 3-D reconstruction and analysis. This method is widely used for 3-D in situ quantitative biology in many areas of biological research. However, the labeling quality and efficiency of standard protocols for fluorescent immunolabeling of these tissue sections are not always satisfactory. Here, we systematically evaluate the effects of raising the conventional antibody incubation temperatures (4°C or 21°C) to mammalian body temperature (37°C) in these protocols. Our modification significantly enhances the quality (labeling sensitivity, specificity, and homogeneity) and efficiency (antibody concentration and antibody incubation duration) of fluorescent immunolabeling of free-floating thick tissue sections.

CD4+ αβ T cell infiltration into the leptomeninges of lumbar dorsal roots contributes to the transition from acute to chronic mechanical allodynia after adult rat tibial nerve injuries

“…Moreover, transverse sections of L4 SC segments were prepared. Representative sections across the whole volume of the target tissues (the sampling ratio as 1:3) were processed for IHC as described previously [ 45 , 46 ]. Coherent parallel processing, appropriate controls, and blind experimental design were used to minimize systematic biases or errors, allowing relatively feasible intergroup comparisons in these and other IHC experiments.…”

“…At the designated time points ( n = 5 animals per group at each time point), transverse frozen sections of L4 SCs (10- or 50-μm thick) from prior lymphadenectomized or sham-operated animals for the LLNs were prepared and processed for fluorescent IHC and imaging as we previously described [ 45 , 46 ]. Free-floating thick sections of L4 SCs before and after mSNIs (five sections per sample) were subjected to GFAP or Iba1 single immunolabeling: mouse monoclonal anti-GFAP (1:5000, Proteintech, 60190-1-Ig; Wuhan, Hubei, China) and rabbit polyclonal anti-Iba1 (1:1000, Proteintech, 10904-1-AP) [ 46 ]. For the quantitative analysis of somatotopically determined IHC staining, the injured tibial innervation territories and the intact sural innervation territories of L4 SC-DH gray matter were determined as previously reported [ 52 ] (Fig.…”

“…Fiji software was used to quantify the MPIs of the positive signal of neuronal PKCγ staining at the inner lamina II of the intact sural innervation territories in L4 SC-DH gray matter [ 52 ]. For both spinal glial and neuronal activation, representative images of almost the same anatomical localization in the matched sections were blindly selected for further processing and the assembly of the figures of this publication [ 46 ].…”

Colocalization of senescent biomarkers in deep, superficial, and ovarian endometriotic lesions: a pilot study

“…To completely understand the machinery of any given cell or tissue and provide accurate molecular assessments, it is pivotal to identify the framework behind its morphology and interactions between its molecules and their compartments 37 . Knowledge of different methods that allow precise and robust evaluation of endometriotic lesions is crucial to address the existing gap on the presentation and etiology of endometriosis and further enhance molecular assessment of endometriotic lesions.…”

TRPV4 and TRPM8 as putative targets for chronic low back pain alleviation

“…After deparaffinization and rehydration through a graded ethanol scale, sections were immersed in 10 mM sodium citrate buffer (pH 6.0) for 10 min in a microwave oven for antigen retrieval and then incubated for 30 min with a blocking solution (2% bovine serum albumin (BSA) and 0.1% Tween20 in phosphate-buffered saline (PBS), 138 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , pH 7.4). Sections were then incubated for 1 h at 37°C [44] with primary antibodies (Alomone Labs, Jerusalem, Israel) all diluted 1:200 in blocking solution. The primary antibodies used are presented in Supplementary Table 1.…”