Antibody Incubation at 37°C Improves Fluorescent Immunolabeling in Free-Floating Thick Tissue Sections

Xiao, Xia; Feng, Yaping; Du, Bin; Sun, Han-Ru; Ding, You-Quan; Qi, Jian-Guo

doi:10.2144/000114524

Cited by 9 publications

(9 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

“…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 ].…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

Ding

Xiao

et al. 2018

J Neuroinflammation

Self Cite

View full text Add to dashboard Cite

Background: Antigen-specific and MHCII-restricted CD4+ αβ T cells have been shown or suggested to play an important role in the transition from acute to chronic mechanical allodynia after peripheral nerve injuries. However, it is still largely unknown where these T cells infiltrate along the somatosensory pathways transmitting mechanical allodynia to initiate the development of chronic mechanical allodynia after nerve injuries. Therefore, the purpose of this study was to ascertain the definite neuroimmune interface for these T cells to initiate the development of chronic mechanical allodynia after peripheral nerve injuries. Methods: First, we utilized both chromogenic and fluorescent immunohistochemistry (IHC) to map αβ T cells along the somatosensory pathways for the transmission of mechanical allodynia after modified spared nerve injuries (mSNIs), i.e., tibial nerve injuries, in adult male Sprague-Dawley rats. We further characterized the molecular identity of these αβ T cells selectively infiltrating into the leptomeninges of L4 dorsal roots (DRs). Second, we identified the specific origins in lumbar lymph nodes (LLNs) for CD4+ αβ T cells selectively present in the leptomeninges of L4 DRs by two experiments: (1) chromogenic IHC in these lymph nodes for CD4+ αβ T cell responses after mSNIs and (2) fluorescent IHC for temporal dynamics of CD4+ αβ T cell infiltration into the L4 DR leptomeninges after mSNIs in prior lymphadenectomized or shamoperated animals to LLNs. Finally, following mSNIs, we evaluated the effects of region-specific targeting of these T cells through prior lymphadenectomy to LLNs and chronic intrathecal application of the suppressive anti-αβTCR antibodies on the development of mechanical allodynia by von Frey hair test and spinal glial or neuronal activation by fluorescent IHC.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Ding

Xiao

et al. 2018

J Neuroinflammation

Self Cite

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Palmieri

Malvezzi

Azevedo

et al. 2022

Sci Rep

View full text Add to dashboard Cite

Endometriosis is a prevalent gynecological condition with deleterious effects on women’s quality of life in terms of physical, emotional, and social compromise. It is an inflammatory disease characterized by the presence of endometrial-like tissue outside the uterus, and its presentation varies from superficial peritoneal lesions to deep infiltrative endometriosis and ovarian endometrioma. In our previous study, endometriotic lesions were implicated in cellular senescence as their inflammatory pattern could potentially compromise surrounding tissue integrity, thereby inducing a senescent state in cells. P16Ink4a and lamin b1 are biomarkers used to assess cellular senescence. Indirect immunofluorescence staining is a broad technique used to assess cellular structure and behavior driven by protein–protein interactions that provide valuable information about cell functioning. The etiopathogeny of endometriosis is not completely understood and diagnostic approaches still rely on invasive methods; therefore, it is important to use validated methods to increase our understanding of the disease and the development of novel diagnostic tools. However, indirect immunofluorescence protocols are often tissue specific and, if neglected, can lead to misinterpretation of results. Moreover, no valid endometriotic tissue-specific colocalization immunofluorescence protocols have been established. Thus, we have validated a well-funded and suitable protocol to allow precise evaluation of the three presentations of endometriosis lesions using indirect immunofluorescence aiming to support further investigations in endometriosis lesions.

show abstract

“…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.…”

Section: Immunofluorescence Analysismentioning

confidence: 99%

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

Fozzato

Baranzini

Bossi

et al. 2020

Pflugers Arch - Eur J Physiol

View full text Add to dashboard Cite

The purpose of this study is to investigate the presence of nervous fibers and expression of TRP channels in samples harvested during decompressive/fusion spine surgeries from patients affected by chronic low back pain (CLBP). The aim was to understand if members of this family of receptors played a role in detection and processing of painful stimuli, to eventually define them as potential targets for CLBP alleviation. Expression of transient receptor potential (TRP) channels (A1, V1, V2, V4, and M8) was evaluated in samples from different periarticular sites of 6 patients affected by CLBP, at both protein and transcript levels. The capsular connective pathological tissue appeared infiltrated by sensitive unmyelinated nervous fibers. An increase in TRP channel mRNAs and proteins was observed in the pathological capsule compared with tissues collected from the non-symptomatic area in five of the six analyzed patients, independently by the location and number of affected sites. In particular, TRPV4 and TRPM8 were consistently upregulated in pathological tissues. Interestingly, the only patient showing a different pattern of expression also had a different clinical history. TRPV4 and TRPM8 channels may play a role in CLBP and warrant further investigations as possible therapeutic targets.

show abstract

Antibody Incubation at 37°C Improves Fluorescent Immunolabeling in Free-Floating Thick Tissue Sections

Cited by 9 publications

References 21 publications

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

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

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

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

Contact Info

Product

Resources

About