Selective Detection of Phagocytic Phase of Apoptosis in Fixed Tissue Sections

2013 ◽  
pp. 159-166 ◽  
Author(s):  
Vladimir V. Didenko
Keyword(s):  
Selective Detection ◽  
Fixed Tissue ◽  
Tissue Sections

Targeted ExSeq -- Tissue Preparation v2

2021 ◽  
Author(s):  
Anubhav Sinha ◽  
Yi Cui ◽  
Shahar Alon ◽  
Asmamaw T. Wassie ◽  
Fei Chen ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Human Tumor ◽  
Metastatic Breast ◽  
Tissue Type ◽  
Model Organisms ◽  
Tissue Preparation ◽  
Enzymatic Reactions ◽  
Fixed Tissue ◽  
Tissue Sections ◽  
Antibody Staining

This protocol accompanies Expansion Sequencing (ExSeq), and describes the tissue preparation for Targeted ExSeq. The steps described here are a generalization of the protocols used for figures 4-6 of the paper, and represent our recommendations for future users of the technology. Fig. 1 shows the structure of the protocol schematically. There are three possible tissue preparation routes described in this protocol that are applicable to different experimental systems. Option (A): harvesting tissue from model organisms that can be transcardially perfused with PFA, followed by sectioning using a vibratome. We typically use this workflow for work on mouse brain sections (see figures 4-5 of ExSeq paper). Option (B): transcardially perfusing with PFA, followed by cryoprotection and cryosectioning. We occasionally use this protocol for work on mouse brain sections. Option (C): snap-freezing fresh tissue (i.e., human tumor biopsy samples, or freshly harvested tissue from mice), followed by cryoprotection and cryosectioning (see figures 2 and 6 of ExSeq paper). The final result of options (A), (B), and (C) is the preparation of fixed tissue sections (either on a glass slide or free-floating). The protocols then briefly converge for optional antibody staining, treatment with LabelX, a chemical that enables anchoring of RNA to the expansion microscopy (ExM) hydrogel, followed by casting of the the ExM gel. There are minor differences in these steps between free-floating and slide-mounted tissue sections, which are noted in the individual steps. The next step, digestion, is tissue-type dependent and may require some optimization for your tissue type. We provide two potential options here: (1) a gentle digestion for tissues such as mouse brain, and (2) a harsh digestion for non-brain tissues such as tumor biopies. The protocols then converge again for the rest of the process. After digestion, the gels are expanded and re-embedded within a second non-expanding hydrogel to lock in the sample size. The carboxylates within the expansion gel are then chemically passivated, enabling enzymatic reactions to be performed within the gel. The samples are now ready for library preparation. In more detail: Steps 1-4 describe the preparation of reagents for downstream steps. The protocol begins either along options (A)/(B), the Transcardial PFA perfusion path (Step 5, continuing to vibratome sectioning in Steps 6-7 for option (A), or cryotome sectioning in Steps 9-10 for option (B)), or along option (C), the Fresh Frozen path (Step 8, continuing to cryotome sectioning in Steps 9-10). The protocols then converge for optional antibody staining (Step 11), followed by LabelX anchoring (Step 12), optional sample trimming (Step 13), and formation of the expansion microscopy gel (Step 14). The details of the digestion step are tissue-type dependent (Step 15). The protocol then concludes with expansion (Step 16), re-embedding (Step 17), passivation, and optional trimming (Steps 18-19). This protocol was used to profile human metastatic breast cancer biopsies as a part of the Human Tumor Atlas Pilot Project (HTAPP). The tissue for this work was collected (see HTAPP-specific tissue collection protocol). The tissue sections were then frozen, cryosectioned, post-fixed, and permeabilized (following steps 9-10). No antibody staining was performed (skipping optional step 11). The sections were then treated with LabelX and gelled (steps 12-14). The gels were then digested using the robust digestion option in steps 15-16. The samples were then re-embedded, passivated, and trimmed (following steps 17-19).

Enhanced DNA fragmentation in the thymus of spontaneously hypertensive rats

1999 ◽  
Vol 276 (6) ◽  
pp. H2135-H2140 ◽  
Author(s):  
Hidekazu Suzuki ◽  
Frank A. Delano ◽  
Neema Jamshidi ◽  
Dan Katz ◽  
Mikiji Mori ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Spontaneously Hypertensive Rat ◽  
Organ Injury ◽  
Fixed Tissue ◽  
Thymic Atrophy ◽  
Spontaneously Hypertensive ◽  
Tissue Sections ◽  
Dna Nicking ◽  
Wistar Kyoto

The mechanisms contributing to organ injury in hypertension have been incompletely defined. The thymus gland of the spontaneously hypertensive rat (SHR) shows significant atrophy at the age of 15 wk compared with its normotensive control, the Wistar-Kyoto rat (WKY). The aim of the present study was to examine the thymus of SHR for evidence of DNA nicking as one of the mechanisms for thymic atrophy. SHR and WKY were subjected to adrenalectomy or sham surgery at 12 wk and studied at 15 wk. Adrenalectomy served to normalize the blood pressure in the SHR. DNA nicking was detected by in situ nick-end labeling (ISEL) of fixed tissue sections. Tissue sections were treated with proteolysis, and terminal deoxyribonucleotidyl transferase was used to incorporate biotinylated deoxynucleotides into DNA nick end in situ. Separately, DNA fragmentation was evaluated by measuring the level of released mono- and oligonucleosomes to the cytoplasm. A higher number of thymic ISEL-positive cells and a higher level of cytoplasmic mono- and oligonucleosomes were observed in SHR than in WKY. After adrenalectomy the enhanced level of ISEL and cytoplasmic mono- and oligonucleosomes in SHR was reduced to the level in WKY. Dexamethasone treatment (0.05 mg ⋅ kg−1⋅ day−1) in WKY serves to decrease the thymus weight and significantly elevate the level of mono- and oligonucleosomes. Thus increased DNA fragmentation represents one of the mechanisms associated with thymic atrophy, a feature that reflects immune suppression in SHR.

In situ transcription: specific synthesis of complementary DNA in fixed tissue sections

Science ◽  
1988 ◽  
Vol 240 (4859) ◽  
pp. 1661-1664 ◽  
Author(s):  
L. Tecott ◽  
J. Barchas ◽  
J. Eberwine
Keyword(s):  
Fixed Tissue ◽  
Complementary Dna ◽  
Tissue Sections ◽  
Specific Synthesis

Effects of antigen retrieval by microwave heating in formalin-fixed tissue sections on a broad panel of antibodies

1994 ◽  
Vol 102 (3) ◽  
pp. 165-172 ◽  
Author(s):  
R. von Wasielewski ◽  
M. Werner ◽  
M. Nolte ◽  
L. Wilkens ◽  
A. Georgii
Keyword(s):  
Microwave Heating ◽  
Antigen Retrieval ◽  
Fixed Tissue ◽  
Formalin Fixed Tissue ◽  
Tissue Sections ◽  
Formalin Fixed

A Basis for Distinguishing Cultured Dendritic Cells and Macrophages in Cytospins and Fixed Sections

2005 ◽  
Vol 8 (1) ◽  
pp. 43-51 ◽  
Author(s):  
Jukka Vakkila ◽  
Michael T. Lotze ◽  
Connie Riga ◽  
Ronald Jaffe
Keyword(s):  
Dendritic Cells ◽  
Cell Types ◽  
Fixed Tissue ◽  
Reliable Identification ◽  
Formalin Fixed Tissue ◽  
Tissue Sections ◽  
Hla Dr ◽  
Formalin Fixed

There is a burgeoning literature on the contrasting role of intratumoral dendritic cells (DCs) and tumor-associated macrophages, making reliable identification of both cell types in clinical and experimental tissue sections important. However, because these cell types are closely related and share several differentiation antigens, their absolute distinction in tissue sections is difficult. We differentiated DCs and macrophages from monocytes in vitro, prepared cytospins and paraffin-embedded sections of the various cell populations, and tested a variety of antibodies that purportedly recognize monocytes and DCs for their capacity to react and distinguish cells after conventional formalin fixation. Cultured DCs but not macrophages were detected by fascin, DC-LAMP, and CD83 with a predictable increase in staining that paralleled their maturation. Staining by CD1a was found on immature DCs but was weak and absent on mature DCs and macrophages, respectively. CD14 and CD163 were characteristic for macrophages and absent on DCs. CD68, HLA-DR, and S100 did not discriminate between DCs and macrophages. We conclude that antigens such as HLA-DR and S100 are not in themselves sufficient for identification of DCs in formalin-fixed tissue sections, but that additional macrophage-specific (CD14, CD163) and DC-specific (CD1a, CD83, fascin, DC-LAMP) antigens should be used to distinguish cell types from each other and to provide information on their state of maturation.

Immunocytochemical localization of RasHa p21 in normal and neoplastic cells in fixed tissue sections from Harvey sarcoma virusinfected mice

1986 ◽  
Vol 7 (4) ◽  
pp. 645-651 ◽  
Author(s):  
Jerrold M. Ward ◽  
Robert L. Pardue ◽  
James L. Junker ◽  
Kiyoshi Takahashi ◽  
Thomas Y. Shih ◽  
...  
Keyword(s):  
Immunocytochemical Localization ◽  
Neoplastic Cells ◽  
Fixed Tissue ◽  
Tissue Sections
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