Journal: Autophagy

Article Title: Immunohistochemical analysis of macroautophagy

doi: 10.4161/auto.22968

Figure Lengend Snippet: Figure 6. Immunohistochemical detection of LC3B is feasible in liver from starved Gfp-Lc3 tg/tg mice using Vectastain ABC. ( A ) Western blot analysis of LC3B in liver from wild-type mice, heterozygous Gfp-Lc3 tg/+ or homozygous Gfp-Lc3 tg/tg transgenic mice. The GFP-LC3 expression in each group was quantified. **p

Article Snippet: Nonspecific protein binding was blocked for 20 min by normal goat serum (Vectastain ABC Kit, Vector Laboratories, PK-4001) or normal horse serum (Vectastain ABC Kit, Vector Laboratories, PK-4002), depending on the animal in which the secondary antibody was raised.

Techniques: Immunohistochemistry, Mouse Assay, Western Blot, Transgenic Assay, Expressing

Journal: Autophagy

Article Title: Immunohistochemical analysis of macroautophagy

doi: 10.4161/auto.22968

Figure Lengend Snippet: Figure 15. SQSTM1 accumulates in cytoplasmic inclusions of liver from autophagy-deficient mice. Liver samples were isolated from fed Atg7 +/+ Alb-Cre + and Atg7 F/F Alb-Cre + mice (control) or from mice that underwent starvation for 48 h. After fixation in neutral buffered formalin for 24 h, tissues were paraffin-embedded and stained for SQSTM1 using rabbit polyclonal anti-SQSTM1 (Sigma, 1:5,000) and Vectastain ABC. Heat-mediated antigen retrieval was performed in citrate buffer (pH 6.0). Scale bar, 20 μm. The positive area of SQSTM1 inclusions was quantified. ***p

Article Snippet: Nonspecific protein binding was blocked for 20 min by normal goat serum (Vectastain ABC Kit, Vector Laboratories, PK-4001) or normal horse serum (Vectastain ABC Kit, Vector Laboratories, PK-4002), depending on the animal in which the secondary antibody was raised.

Techniques: Mouse Assay, Isolation, Staining

Journal: Autophagy

Article Title: Immunohistochemical analysis of macroautophagy

doi: 10.4161/auto.22968

Figure Lengend Snippet: Figure 5. The highest expression of LC3A and LC3B in nonstarved control mice was found in brain tissue. ( A ) Western blot analysis of LC3A and LC3B in different tissue lysates. GAPDH served as a loading control. ( B ) Immunohistochemical detection of LC3A and LC3B in mouse brain using the Vectastain ABC system. Tissue samples were isolated from fed control mice. After fixation in neutral buffered formalin for 24 h, tissues were paraffin-embedded and stained for LC3A ( A ) and LC3B ( B ) using rabbit polyclonal anti-LC3A (Abgent, 1:3000) and biotinylated mouse monoclonal anti-LC3B (clone 5F10, Nanotools, 1:100). Heat-mediated antigen retrieval was performed in citrate buffer (pH 6.0). Scale bar, 20 μm.

Article Snippet: Nonspecific protein binding was blocked for 20 min by normal goat serum (Vectastain ABC Kit, Vector Laboratories, PK-4001) or normal horse serum (Vectastain ABC Kit, Vector Laboratories, PK-4002), depending on the animal in which the secondary antibody was raised.

Techniques: Expressing, Mouse Assay, Western Blot, Immunohistochemistry, Isolation, Staining

Journal: Autophagy

Article Title: Immunohistochemical analysis of macroautophagy

doi: 10.4161/auto.22968

Figure Lengend Snippet: Figure 13. ATG5 and CTSD are not suitable targets for the immunohistochemical detection of autophagy in liver. Liver samples were isolated from fed Atg7 +/+ Alb-Cre + and Atg7 F/F Alb-Cre + mice (control) or from mice that underwent starvation for 48 h. After fixation in neutral buffered formalin for 24 h, tissues were paraffin-embedded and stained for ATG5 ( A ) or CTSD ( B ) using rabbit polyclonal anti-ATG5 (Abcam, 1:100) and rabbit monoclonal anti-CTSD (clone EPR3057Y, Abcam, 1:1000), respectively, in combination with Vectastain ABC. Heat-mediated antigen retrieval was performed in citrate buffer (pH 6.0). Scale bar, 20 μm. The ATG5- and CTSD-positive area was quantified. Neither the effect of starvation nor the results between Atg7 +/+ Alb-Cre + and Atg7 F/F Alb-Cre + mice were statistically significant.

Article Snippet: Nonspecific protein binding was blocked for 20 min by normal goat serum (Vectastain ABC Kit, Vector Laboratories, PK-4001) or normal horse serum (Vectastain ABC Kit, Vector Laboratories, PK-4002), depending on the animal in which the secondary antibody was raised.

Techniques: Immunohistochemistry, Isolation, Mouse Assay, Staining

Journal: Autophagy

Article Title: Immunohistochemical analysis of macroautophagy

doi: 10.4161/auto.22968

Figure Lengend Snippet: Figure 7. Optimal immunohistochemical detection of LC3B requires processing of tissue samples in a suitable fixative. Liver samples were isolated from Gfp-Lc3 tg/tg mice that underwent starvation for 48 h. After fixation in different fixatives for 24 h, tissues were paraffin-embedded and stained for LC3B using biotinylated mouse monoclonal anti-LC3B (clone 5F10, Nanotools, 1:100) and Vectastain ABC. For formalin-fixed samples, heat-mediated antigen retrieval was performed either in citrate buffer (pH 6.0) or in EDTA buffer (pH 8.0). Scale bar, 20 μm.

Article Snippet: Nonspecific protein binding was blocked for 20 min by normal goat serum (Vectastain ABC Kit, Vector Laboratories, PK-4001) or normal horse serum (Vectastain ABC Kit, Vector Laboratories, PK-4002), depending on the animal in which the secondary antibody was raised.

Techniques: Immunohistochemistry, Isolation, Mouse Assay, Staining

Journal: Autophagy

Article Title: Immunohistochemical analysis of macroautophagy

doi: 10.4161/auto.22968

Figure Lengend Snippet: Figure 4. Liver from autophagy-deficient Atg7 F/F Alb-Cre + mice but not from autophagy-competent Atg7 +/+ Alb-Cre + shows immunohistochemical staining for LC3A and LC3B. Liver samples were isolated from fed mice (control) or from mice that underwent starvation for 48 h. After fixation in neutral buffered formalin for 24 h, tissues were paraffin-embedded and stained for LC3A ( A ) and LC3B ( B ) using rabbit polyclonal anti-LC3A (Abgent, 1:100) and biotinylated mouse monoclonal anti-LC3B (clone 5F10, Nanotools, 1:100) in combination with Vectastain ABC. Heat-mediated antigen retrieval was performed in citrate buffer (pH 6.0). Scale bar, 20 μm. The LC3A and LC3B positive area was quantified. ***p

Article Snippet: Nonspecific protein binding was blocked for 20 min by normal goat serum (Vectastain ABC Kit, Vector Laboratories, PK-4001) or normal horse serum (Vectastain ABC Kit, Vector Laboratories, PK-4002), depending on the animal in which the secondary antibody was raised.

Techniques: Mouse Assay, Immunohistochemistry, Staining, Isolation

Journal: Autophagy

Article Title: Immunohistochemical analysis of macroautophagy

doi: 10.4161/auto.22968

Figure Lengend Snippet: Figure 9. LC3B dots are detectable in frozen liver sections using a staining procedure with alkaline phosphatase and are localized on the surface of lipid droplets. Liver samples were isolated from Atg7 +/+ Alb-Cre + ( A ) or Atg7 F/F Alb-Cre + mice ( B ) that were fed normal chow (control) or underwent starvation for 48 h. After fixation in 4% paraformaldehyde, frozen sections were stained for LC3B using biotinylated mouse monoclonal anti-LC3B (clone 5F10, Nanotools, 1:1,000) and Vectastain ABC-AP kit, containing biotinylated alkaline phosphatase instead of biotinylated horseradish peroxidase. LC3B-positive dots (arrows) were detected on the surface of lipid droplets. These structures could be stained using oil red O. Scale bar, 20 μm.

Article Snippet: Nonspecific protein binding was blocked for 20 min by normal goat serum (Vectastain ABC Kit, Vector Laboratories, PK-4001) or normal horse serum (Vectastain ABC Kit, Vector Laboratories, PK-4002), depending on the animal in which the secondary antibody was raised.

Techniques: Staining, Isolation, Mouse Assay

Journal: Autophagy

Article Title: Immunohistochemical analysis of macroautophagy

doi: 10.4161/auto.22968

Figure Lengend Snippet: Figure 8. Immunohistochemical staining of LC3B is enhanced in frozen liver sections. Liver samples were isolated from Atg7 +/+ Alb-Cre + and Atg7 F/F Alb-Cre + mice ( A ) or from transgenic Gfp-Lc3 tg/+ or Gfp-Lc3 tg/tg mice ( B ). Some animals were fed normal chow (control), others underwent starvation for 48 h. After fixation in acetone, frozen sections were stained for LC3B using biotinylated mouse monoclonal anti-LC3B (clone 5F10, Nanotools, 1:1,000 [ Atg7 +/+ Alb-Cre + and Atg7 F/F Alb-Cre + samples] or 1:30,000 [ Gfp-Lc3 tg/+ and Gfp-Lc3 tg/tg samples]) and Vectastain ABC. Scale bar, 40 μm.

Article Snippet: Nonspecific protein binding was blocked for 20 min by normal goat serum (Vectastain ABC Kit, Vector Laboratories, PK-4001) or normal horse serum (Vectastain ABC Kit, Vector Laboratories, PK-4002), depending on the animal in which the secondary antibody was raised.

Techniques: Immunohistochemistry, Staining, Isolation, Mouse Assay, Transgenic Assay

Journal: Autophagy

Article Title: Immunohistochemical analysis of macroautophagy

doi: 10.4161/auto.22968

Figure Lengend Snippet: Figure 14. BECN1 is upregulated in mouse tissue after starvation. ( A ) Liver samples were isolated from fed Atg7 +/+ Alb-Cre + and Atg7 F/F Alb-Cre + mice (control) or from mice that underwent starvation for 48 h. After fixation in neutral buffered formalin for 24 h, tissues were paraffin-embedded and stained for BECN1 using rabbit polyclonal anti-BECN1 (Lifespan, 1:1,000) and Vectastain ABC. Heat-mediated antigen retrieval was performed in citrate buffer (pH 6.0). Scale bar 20 μm. The BECN1 positive area was quantified. ***p

Article Snippet: Nonspecific protein binding was blocked for 20 min by normal goat serum (Vectastain ABC Kit, Vector Laboratories, PK-4001) or normal horse serum (Vectastain ABC Kit, Vector Laboratories, PK-4002), depending on the animal in which the secondary antibody was raised.

Techniques: Isolation, Mouse Assay, Staining